Bicyclic compounds and methods for their use in treating autism

ABSTRACT

Embodiments of this invention provide compositions and methods for therapeutic use of diketopiperazines including cyclic G-2-Allyl Proline and other cyclic Glycyl Proline compounds to treat symptoms of Autistic Disorder or Autism, as well as manufacture of medicaments including tablets, capsules, liquid formulations, gels, injectable solutions, and other formulations that are useful for treatment of such conditions.

CLAIM OF PRIORITY

This United States Continuation application is filed under 35 U.S.C. §111a, claims priority to U.S. patent application Ser. No. 15/869,672filed 12 Jan. 2018 (U.S. Pat. No. 10,548,892, issued 4 Feb. 2020), whichclaims priority to U.S. patent application Ser. No. 15/004,218 filed 22Jan. 2016 (now U.S. Pat. No. 9,867,823, issued 16 Jan. 2018, which is acontinuation of International Patent Application No. PCT/US2014/047801,filed Jul. 23, 2014, entitled “Neuroprotective Bicyclic Compounds andMethods for Their Use in Treating Autism Spectrum Disorders andNeurodevelopmental Disorders,” Inventors Lawrence Irwin Glass, MichaelJohn Bickerdike, Michael Fredrick Snape, and Patricia Perez de Cogram,which claims priority to U.S. Provisional Patent Application No.61/958,329 filed 25 Jul. 2013 entitled “Neuroprotective BicyclicCompounds and Methods for Their Use in Treating Autism SpectrumDisorders and Neurodevelopmental Disorders,” Inventors Lawrence IrwinGlass, Michael John Bickerdike, Michael Fredrick Snape, and PatriciaPerez de Cogram. Each of these patents and patent applications areincorporated herein fully by reference.

FIELD OF THE INVENTION

The present invention relates to novel bicyclic compounds structurallyrelated to diketopiperazines and methods for their therapeutic use. Inparticular, this invention relates to the neuroprote active activity ofsuch compounds. More particularly, this invention relates to the use ofcyclic Glycyl Proline (“cPG”) and analogs thereof, including cyclicGlycyl-2-Allyl Proline (“cyclic G-2-AllylP” or “cG-2-AllylP” or “NNZ2591”) and pharmaceutical compositions thereof in the treatment ofNeurodevelopmental Disorders, and particularly directed to treatment ofAutistic Disorder and autism.

BACKGROUND

Autism Spectrum Disorders (ASDs) are increasingly being diagnosed. ASDsare a collection of linked developmental disorders, characterized byabnormalities in social interaction and communication, restrictedinterests, and repetitive behaviours. In addition to classical autism orAutistic Disorder, the fifth edition of the American PsychiatricAssociation's (APA) Diagnostic and Statistical Manual of MentalDisorders (DSM-5) recognizes Autistic Disorder, Asperger syndrome,Childhood Disintegrative Disorder, and Pervasive Developmental DisorderNot Otherwise Specified (PDD-NOS) as ASDs.

Neurodevelopmental Disorders (NDDs) include Fragile X Syndrome (FXS),Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome,Rett Syndrome, CDKL5 mutations (which also are associated with RettSyndrome and X-Linked Infantile Spasm Disorder) and others. Many but notall NDDs are caused by genetic mutations and, as such, are sometimesreferred to as monogenic disorders. Some patients with NDDs exhibitbehaviors and symptoms of autism.

As an example of a NDD, Fragile X Syndrome is an X-linked geneticdisorder in which affected individuals are intellectually handicapped tovarying degrees and display a variety of associated psychiatricsymptoms. Clinically, Fragile X Syndrome is characterized byintellectual handicap, hyperactivity and attentional problems, autismspectrum symptoms, emotional lability and epilepsy (Hagerman, 1997a).The epilepsy seen in Fragile X Syndrome is most commonly present inchildhood, but then gradually remits towards adulthood. Hyperactivity ispresent in approximately 80 percent of affected males (Hagerman, 1997b).Physical features such as prominent ears and jaw and hyper-extensibilityof joints are frequently present but are not diagnostic. Intellectualhandicap is the most common feature defining the phenotype. Generally,males are more severely affected than females. Early impressions thatfemales are unaffected have been replaced by an understanding of thepresence of specific learning difficulties and other neuropsychiatricfeatures in females. The learning disability present in males becomesmore defined with age, although this longitudinal effect is more likelya reflection of a flattening of developmental trajectories rather thanan explicit neurodegenerative process.

The compromise of brain function seen in Fragile X Syndrome isparalleled by changes in brain structure in humans. MRI scanning studiesreveal that Fragile X Syndrome is associated with larger brain volumesthan would be expected in matched controls and that this changecorrelates with trinucleotide expansion in the FMRP promoter region(Jakala et al., 1997). At the microscopic level, humans with Fragile XSyndrome show abnormalities of neuronal dendritic structure, inparticular, an abnormally high number of immature dendritic spines(Irwin et al., 2000).

Currently available treatments for NDDs are symptomatic—focusing on themanagement of symptoms—and supportive, requiring a multidisciplinaryapproach. Educational and social skills training and therapies areimplemented early to address core issues of learning delay and socialimpairments. Special academic, social, vocational, and support servicesare often required. Medication, psychotherapy or behavioral therapy maybe used for management of co-occurring anxiety, Attention DeficitHyperactivity Disorder (“ADHD”), depression, maladaptive behaviors suchas aggression, and sleep issues. Antiepileptic drugs may be used tocontrol seizures.

SUMMARY

We have previously shown in patent application PCT/US2004/02830 filedAug. 31, 2004, expressly incorporated herein fully by reference, thatcyclic Glycyl Proline (“cPG”) and analogues thereof, including but notlimited to cyclic cyclopentyl-G-2-MeP and cyclic-G-2-AllylP )“cG-2-AllylP”) are neuroprotective and neuroregenerative. There is nocurrent, effective treatment of ASDs or NDDs, and patient care islimited to management of the symptoms, predominantly using social andbehavioural interventions. The inventors have now discovered that cyclicG-2-AllylP and other bicyclic compounds disclosed herein can beeffective in treatment of ASDs and NDDs, and in particular fornormalizing abnormal social behavior.

We unexpectedly discovered that cG-2-AllylP has a robust therapeuticeffect on anxiety, hyperactivity, memory, learning and species-typicalabnormal social behaviour and repetitive behavior in animals havingFragile X Syndrome (“FXS”) or other ASDs. In addition, we found thatchanges in ERK1/2 and Akt phosphorylation in cG-2-AllylP treatedfmr1-knockout animals, provides an in vitro diagnostic evaluation ofASDs, and support the hypothesis that the phenotypes of Fragile XSyndrome are the result of altered mGluR expression. Moreover,administration of cG-2-AllylP significantly reduced the numbers ofneuronal spines in fmr1-knockout mice.

Because the fmr1-knockout animals used for in vivo studies disclosedherein have the same genetic mutation as human beings with Fragile XSyndrome, administration of cyclic Glycyl Proline (“cGP”) compounds ofthis invention, including cG-2-AllylP, can be useful in treatingsymptoms of Autism Spectrum Disorders, Neurodevelopmental Disorders andFragile X Syndrome in human beings. In addition, we unexpectedly foundthat cGPs of this invention can effectively treat adverse socialbehaviors and repetitive behaviors in animals with ASDs, thus restoringmore normal social interactions.

Thus, one aspect of this invention provides novel cyclic compoundshaving the structural formulas and substituents described below.

In some aspects, compounds of Formula 1 include substituents where:

X¹ is selected from the group consisting of NR′, O and S;

X² is selected from the group consisting of CH₂, NR′, O and S;

R¹, R², R³, R⁴ and R⁵ are independently selected from the groupconsisting of —H, —OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′,—C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl, halogen, alkyl, substitutedalkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl,heteroarylalkyl and substituted heteroarylalkyl; each R′ isindependently selected from the group consisting of —H, alkyl,heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl; or R⁴ and R⁵ taken together are —CH₂—(CH₂)_(n)—CH₂—where n is an integer from 0-6; or R² and R³ taken together are—CH₂—(CH₂)_(n)—CH₂— where n is an integer from 0-6; with the provisothat when R¹=methyl and R²=R³=R⁴=H then R⁵≠benzyl and; when R¹=H, atleast one of R² and R³≠H.

In further aspects, this invention provides a compound of Formula 1 or apharmaceutically acceptable salt, stereoisomer or hydrate thereof,wherein R¹=allyl, R²=R³=R⁴=R⁵=H, X¹=NH, X²=CH₂ (cyclicGlycyl-2-AllylProline).

In still other aspects, this invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of cyclic G-2AllylP.

In further aspects, this invention provides methods of treating ananimal having a cognitive impairment, comprising administration to thatanimal an effective amount of a composition comprising cyclicG-2-AllylP. In yet further aspects, the animal to be treated is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Color Drawings:

This application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

This invention is described with reference to specific embodimentsthereof. Other aspects of this invention can be appreciated withreference to the drawings, in which:

FIG. 1A is a graph showing effects of treatment with cyclic G-2-AllylPon the performance in acquisition phase (days 1-4) of the Morris WaterMaze Test (MWMT) following scopolamine treatment.

FIG. 1B is a graph showing effects of treatment with cyclic G-2-AllylPon the latency to the platform quadrant in the probe test (day 5) of theMWMT.

FIG. 1C is a graph showing the time taken to find the platform on day4^(th) of the acquisition phase for animals in 3 groups: (1)vehicle-treated, (2) scopolamine and cG-2-AllylP-treated and (3)scopolamine-treated.

FIG. 2 is a graph showing the difference in time spent on exploring thefamiliar vs novel object during the probe test on days 25post-treatment. The data points for familiar objects reflect the averageof time spent on exploration of 3 familiar objects. The data point fornovel object recognition is the actual time spent exploring the novelobject.

FIG. 3 is a graph showing a correlation between the AMPA glutamatereceptor-1 staining of the CA1 region of the hippocampus and the ratioof time spend on investigation of novel object to familiar object intesting phase of the NORT on day 24.

FIG. 4 is a graph showing the effects of cG-2-AllylP (t) on the densityof AMPA GluR1 in CA1 granular cell layer on days 6 and 24 in comparisonto vehicle (veh).

FIG. 5 is a graph showing the effects of cG-2-AllylP (t) on the densityof AMPA GluR1 in CA1 stratum oriens on day 24 post treatment.

FIG. 6 is a graph showing the effect of cG-2-AllylP on the trend toincrease the density of pre-synaptic stain in CA3 region of thehippocampus at day 24 post-treatment.

FIG. 7 is a graph showing the effect of cG-2-AllylP on the trend toincrease the density of the pre-synaptic stain in the stratum oriens ofthe CA1 region on day 24 post-treatment.

FIG. 8 is a graph showing the effect of cG-2-AllylP to increase thedensity of the pre-synaptic stain in the stratum radiatum of the CA1region on day 24 post-treatment.

FIGS. 9A, 9B, and 9C are graphs showing the effect of cG-2-AllylPtreatment on the density of the NMDAR-1 in CA1 and CA3, respectively.

FIG. 10 is a graph showing the effects of cG-2-AllylP on the density ofKrox24 staining in the CA1-2 of the hippocampus.

FIG. 11 is a graph showing the effects of cG-2-AllylP on the number ofvehicles in a 200 nm² square apposing the post-synaptic density insubregions CA3 and CA1 of the hippocampus of middle aged rats (n=2 ineach group).

FIG. 12 is a graph showing effects of cyclic G-2-AllylP on neuronalsurvival in animals following excitotoxic oxidative stress.

FIG. 13 is a graph showing effects of cyclic cyclopentylG-2-MeP onneuronal survival in animals following excitotoxic oxidative stress.

FIG. 14 is a graph showing the neuroprotective effects of cyclicG-2-AllylP in animals subjected to global brain ischaemia.

FIG. 15 is a graph showing effects of different doses of cyclicG-2-AllylP on neuroprotection in animals subjected to global brainischaemia.

FIGS. 16A-D depict a chamber for studies of hippocampal neurons.

FIG. 16A depicts a chamber used for in vitro studies of hippocampalneurons.

FIG. 16B depicts a photograph of hippocampal neurons after 17 days inculture.

FIG. 16C depicts GFP labelled Fmr1 knockout hippocampal neurons.

FIG. 16D depicts a photograph of hippocampal neurons from Fmr1 knockoutmice treated with cG-2-2AllylP.

FIG. 17 depicts a photograph of an Open Field Test device used to testeffects of cG-2-AllylP of this invention.

FIG. 18 depicts graph of the time T1 spent in Open Field Test of wildtype animals and fmr1 knockout animals treated with either vehicle orcG-2-AllylP.

FIG. 19 depicts a graph of results of short-tem memory in an Open FieldTest of wild-type animals and fmr1-knockout animals treated with eithervehicle or cG-2-AllylP.

FIG. 20 depicts a graph of results of long-tem memory in an Open FieldTest of wild-type animals and fmr1-knockout animals treated with eithervehicle or cG-2-AllylP.

FIG. 21 depicts a graph of results of a Successive Alleys Test inwild-type animals and fmr1-knockout animals treated with vehicle.

FIG. 22 depicts a graph of results of a Successive Alleys Test inwild-type animals and fmr1-knockout animals treated with cG-2-AllylP.

FIG. 23 depicts a photograph of an Elevated Plus Maze used in studies ofeffects of cG-2-AllylP of this invention in wild-type mice andfmr1-knockout mice.

FIG. 24 depicts a graph of results in an Elevated plus Maze Closed ArmTest in wild-type animals and fmr1-knockout animals treated with eithervehicle or cG-2-AllylP.

FIG. 25 depicts a graph of results in an Elevated Plus Maze Open ArmTest in wild-type animals and fmr1-knockout animals treated with eithervehicle or cG-2-AllylP.

FIG. 26 depicts a graph of results in an Elevated Plus Maze Center Testin wild-type animals and fmr1-knockout animals treated with eithervehicle or cG-2-AllylP.

FIG. 27 depicts a photograph of a device used to study effects ofcG-2-AllylP on fear conditioning in wild-type and fmr1-knockout mice.

FIG. 28 depicts a graph of results in a Fear Conditioning Test inwild-type animals and fmr1-knockout animals treated with either vehicleor cG-2-AllylP.

FIGS. 29A-E depict photographs of nesting scores used in evaluatingeffects of cG-2-AllylP on nesting behavior in wild-type mice andfmr1-knockout mice.

FIG. 29A depicts score of 1.

FIG. 20B depicts a score of 2.

FIG. 29C depicts a score of 3.

FIG. 20D depicts a score of 4.

FIG. 29E depicts a score of 5.

FIG. 30 depicts a graph of results in a Sociability Test in wild-typeanimals and fmr1-knockout animals treated with either vehicle orcG-2-AllylP.

FIG. 31 depicts a graph of results expression levels of pERK inwild-type animals and fmr1-knockout animals treated with either vehicleor cG-2-AllylP.

FIG. 32 depicts a graph of results of expression levels of pAKT inwild-type animals and fmr1-knockout animals treated with either vehicleor cG-2-AllylP.

FIGS. 33A, 33B, 33C, 33D, 33E, and 33F depict graphs of results ofeffects of PBBI and cG-2-AllylP on expression of inflammatory mediatorsinterleukin 1-beta (“IL1-beta”) and interleukin 6 (“IL-6”).

FIGS. 33A, 33B, and 33C depict results for IL1-beta and

FIGS. 33D, 33E, and 33F depict results for IL-6.

FIG. 34A, 34B, 34C, 34D, 34E, 34F, 34 g, and 34 H depict graphs ofresults of effects of PBBI and cG-2-AllylP on expression of BAX andBCL-2.

FIGS. 34A, 34B, and 34C depict results for BAX expression.

FIGS. 34D, 34E, 34F, 34G, and 34H depict results for BCL2 expression.

FIGS. 35A, 34B, and 35C depicts graphs of results of effects of PBBI andcG-2-AllylP on expression of ATF3 at three different time points.

FIG. 36A through 36O depict graphs of results of effects of PBBI andcG-2-AllylP on expression of several genetic markers related toneuroplasticity. FIG. 36A depicts a graph of results of effects of PBBIand cG-2-AllylP on expression of BDNF. FIG. 36B depicts a graph ofresults of effects of PBBI and cG-2-AllylP on expression of Cdh2. FIG.36C depicts a graph of results of effects of PBBI and cG-2-AllylP onexpression of Cebpb. FIG. 36D depicts a graph of results of effects ofPBBI and cG-2-AllylP on expression of Crem. FIG. 36E depicts a graph ofresults of effects of PBBI and cG-2-AllylP on expression of Egr1. FIG.36F depicts a graph of results of effects of PBBI and cG-2-AllylP onexpression of Gria4. FIG. 36G depicts a graph of results of effects ofPBBI and cG-2-AllylP on expression of Grm5. FIG. 36H depicts a graph ofresults of effects of PBBI and cG-2-AllylP on expression of Mapk1. FIG.36I depicts a graph of results of effects of PBBI and cG-2-AllylP onexpression of Nt4a1. FIG. 36J depicts a graph of results of effects ofPBBI and cG-2-AllylP on expression of Ntf3. FIG. 36K depicts a graph ofresults of effects of PBBI and cG-2-AllylP on expression of Ntf4. FIG.36L depicts a graph of results of effects of PBBI and cG-2-AllylP onexpression of Pcdh8. FIG. 36M depicts a graph of results of effects ofPBBI and cG-2-AllylP on expression of Pim1. FIG. 36N depicts a graph ofresults of effects of PBBI and cG-2-AllylP on expression of Ppp3ca. FIG.36O depicts a graph of results of effects of PBBI and cG-2-AllylP onexpression of Tnf.

DETAILED DESCRIPTION Definitions

“Alkenyl” refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon double bond. Theradical may be in either the cis or trans conformation about the doublebond(s). Exemplary alkenyl groups include allyl, ethenyl, propenyl,isopropenyl, butenyl, isobutenyl, cyclopentenyl and the like. In someembodiments the alkenyl groups are (C₂-C₆) alkenyl, and in otherembodiments, allyl can be particularly useful.

“Alkyl” refers to a saturated branched, straight chain or cyclichydrocarbon radical. Exemplary alkyl groups include methyl, ethyl,isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, hexyl and thelike. In some embodiments the alkyl groups are (C₁-C₆) alkyl.

“Alkynyl” refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon triple bond.Exemplary alkynyl groups include ethynyl, propynyl, butynyl, isobutynyland the like. In some embodiments the alkynyl group is (C₂-C₆) alkynyl.

“Aryl” refers to an unsaturated cyclic hydrocarbon radical with aconjugated π electron system. Exemplary aryl groups include phenyl,naphthyl and the like. In some embodiments the aryl group is (C₃-C₂₀)aryl.

“Arylalkyl” refers to a straight chain alkyl, alkenyl or alkynyl groupwherein one of the hydrogen atoms bound to the terminal carbon isreplaced with an aryl group. Exemplary arylalkyl groups include benzyl,naphthylmethyl, benzylidene and the like.

Cognitive impairment can be observed in patients having ASDs, NDDs,Alzheimer's disease, Parkinson's disease, Lewy-bodies dementia and otherdisorders, as well in aging animals, including humans.

“Comprising,” and “Comprises” means including, but not limited to theelements listed.

“Growth factor” refers to an extracellularly active polypeptide thatstimulates a cell to grow or proliferate by interacting with a receptoron the cell.

“Heteroalkyl” refers to an alkyl moiety wherein one or more carbon atomsare replaced with another atom such as N, P, O, S etc. Exemplaryheteroalkyl groups include pyrrolidine, morpholine, piperidine,piperazine, imidazolidine, pyrazolidine, tetrahydrofuran, (C₁-C₁₀)substituted amines, (C₂-C₆) thioethers and the like.

“Heteroaryl” refers to an aryl moiety wherein one or more carbon atomsare replaced with another atom such as N, P, O, S etc. Exemplaryheteroaryl groups include carbazole, furan, imidazole, indazole, indole,isoquinoline, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrrole,thiazole, thiophene, triazole and the like.

“Injury” includes any acute or chronic damage of an animal that resultsin degeneration, dysfunction or death of cells in the nervous system.Such cells include neuronal cells and non-neuronal cells. Injuryincludes stroke, non-hemorrhagic stroke, traumatic brain injury,perinatal asphyxia associated with fetal distress such as followingabruption, cord occlusion or associated with intrauterine growthretardation, perinatal asphyxia associated with failure of adequateresuscitation or respiration, severe CNS insults associated with nearmiss drowning, near miss cot death, carbon monoxide inhalation, ammoniaor other gaseous intoxication, cardiac arrest, coma, meningitis,hypoglycaemia, status epilepticus, episodes of cerebral asphyxiaassociated with coronary bypass surgery, hypotensive episodes andhypertensive crises, and cerebral trauma. It is to be understood thatthe above examples are by way of illustration only, and are not intendedto be a complete listing of injuries capable of being treated by thecompounds and methods of this invention.

A “pharmaceutically acceptable excipient” refers to an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

A “pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable and has the desired pharmacologicalproperties. Such salts include salts that may be formed where acidicprotons present in the compounds are capable of reacting with inorganicor organic bases. Suitable inorganic salts include those formed with thealkali metals, e.g. sodium and potassium, magnesium, calcium, andaluminium. Suitable organic salts include those formed with organicbases such as the amine bases e.g. ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like. Suchsalts also include acid addition salts formed with inorganic acids (e.g.hydrochloric and hydrobromic acids) and organic acids (e.g. acetic acid,citric acid, maleic acid, and the alkane- and arene-sulfonic acids suchas methanesulfonic acid and benzenesulfonic acid). When there are twoacidic groups present, a pharmaceutically acceptable salt may be amono-acid mono-salt or a di-acid salt; and similarly where there aremore than two acidic groups present, some or all of such groups can bepresent as salts.

A “protecting group” has the meaning conventionally associated with itin organic synthesis, i.e. a group that selectively blocks one or morereactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete.

A “stereoisomer” is a molecule having the structure of cyclic G-2-AllylProline, but having a chiral center. The term “cyclic G-2-Allyl Proline”includes all stereoisomers.

“Substituted” refers to where one or more of the hydrogen atoms on analkyl, heteroalkyl, alkenyl, alkynyl, aryl, heteroaryl or arylalkylradical are independently replaced with another substituent.Substituents include —R′, —OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′,—C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, —NR′—C(NR′)—OR′, —NR′—C(NR′)—SR′,NR′—C(NR′NR′R′, trihalomethyl and halogen where each R′ is independently—H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl orheteroarylalkyl.

“Symptom” or “symptoms” means one or more of cognitive impairment orcognitive dysfunction, one or more signs or symptoms of memory loss,loss of spatial orientation, decreased ability to learn, decreasedability to form short- or long-term memory, decreased episodic memory,decreased ability to consolidate memory, decreased spatial memory,decreased synaptogenesis, decreased synaptic stability, deficits inexecutive function, deficits in cognitive mapping and scene memory,deficits in declarative and relational memory, decreased rapidacquisition of configural or conjunctive associations, decreasedcontext-specific encoding and retrieval of specific events, decreasedepisodic and/or episodic-like memory, anxiety, abnormal fearconditioning, abnormal social behaviour, repetitive behaviour, abnormalnocturnal behavior, seizure activity, abnormal locomotion, abnormalexpression of Phospho-ERK1/2 and Phospho-Akt, and bradycardia.

A “therapeutically effective amount” means the amount that, whenadministered to an animal for treating a disease, is sufficient toeffect treatment for a disease or an injury. A “therapeuticallyeffective amount” means an amount that decreases adverse symptoms orfindings, promotes desirable symptoms or findings, and/or treats anunderlying disorder, and/or is curative.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in an animal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease).

Implicit hydrogen atoms (such as the hydrogens on the pyrrole ring,etc.) are omitted from the formulae for clarity, but should beunderstood to be present.

“ATF3” means Activating Transcription Factor 3,

“BAX” means the apoptosis regulator BAX also known as bcl-2-likeprotein,

“BLC2 alpha” means the B-cell lymphoma-2,

“IL1-beta” means Interleukin 1-beta.

“IL-6” means Interleukin-6.

“BDNF” means Brain Derived Neurotropic factor.

“Cdh2” means Cadherin-2,

“Cebpb” means CCAAT/enhancer-binding protein beta,

“Crem” means cyclic-AMP response element binding,

“Egr1” means Early Growth Response Protein 1,

“Gria 4” means Glutamate Receptor Ionotropic AMPA 4,

“Grm5” means Metabotropic Glutamate Receptor S,

“Mapk 1” means Mitogen-Activated Protein Kinase 1,

“MeCP2” means Methyl cPg Binding Protein 2,

“Nr4a1” means Nuclear Receptor Subfamily 4 Group A member 1, also knownas Nerve Growth Factor IB,

“Ntf3” means Neurotrophin 3,

“Ntf4” means Neurotrophin 4,

“Pcdh8” means Protocadherin-8,

“Plm1” means Pre-mRNA Leakage Protein 1,

“Ppp3ca” means Protein Phosphatase 3, Catalytic Subunit, Alpha.

“Tnf” means Tumor Necrosis Factor.

Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a collection of linkeddevelopmental disorders, characterized by abnormalities in socialinteraction and communication, restricted interests and repetitivebehaviours. In addition to classical Autism or Autistic Disorder, thefifth edition of the American Psychiatric Association's (APA) Diagnosticand Statistical Manual of Mental Disorders (DSM-3) recognises Aspergersyndrome. Childhood Disintegrative Disorder and Pervasive DevelopmentalDisorder Not Otherwise Specified (PDD-NOS) as ASDs.

Neurodevelopmental Disorders (NDDs) include Fragile X Syndrome (FXS),Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome,Rett Syndrome, CDKL5 mutations (which also are associated with RettSyndrome and X-Linked Infantile Spasm Disorder) and others. Many but notall NDDs are caused by genetic mutations and, as such, are sometimesreferred to as monogenic disorders. Some patients with NDDs exhibitbehaviors and symptoms of autism.

Clinical Tools for Evaluating ASDs and NDDs

ASDs and NDDs can be assessed using one or more clinical tests, forexample. The Rett Syndrome Natural History/Clinical Severity Scale,Aberrant Behavior Checklist Community Edition (ABC), Aberrant BehaviorChecklist (Stereotypy), Vinelands, Clinical Global Impression ofSeverity (CGI-S), the Caregiver Strain Questionnaire (CSQ), Children'sYale-Brown OC Scale (CYBOCS-PDD), Child Autism Rating Scale, Interviewof Repetitive Behaviors, Nisonger Child Behavior Rating Scale, PervasiveDevelopmental Disorder Behav Inventory, Stereotyped Behav Scale,Repetitive Behavior Scale, Rossago Scale, Repetitive BehaviorQuestionnaire, and Stereotyped Behavior Scale, or one or morephysiological test selected from the group consisting ofelectroencephalogram (EEG) spike frequency, overall power in frequencybands of an EEG, hand movement, QTc and heart rate variability (HRV),and respiratory irregularities compared to control animals not sufferingfrom said disorder. Reliability and relevance of some of these tools asshown in Table 1 below.

As used in this section, the term “Appropriate” means a tool thatmeasures a clinically relevant outcome with good to excellentreliability and validity with information available on all relevantcategories. The term “Appropriate with Conditions” means a tool thatmeasures a “clinically relevant outcome for which only certain subscalesare relevant,” or may be “only relevant for younger age range.” Theterms “Promising” and Potentially Appropriate” mean a tool that measures“a clinically relevant outcome but is emerging, or has inconsistentreliability and validity (e.g., good to excellent reliability/validitybut data is not available in all categories, but a least 2, adequate inall categories.”

TABLE 1 Clinical Tools Considered “Appropriate” or “Appropriate withConditions” for Restricted and Repetitive Behaviors in ASDs and NDDsReliable Sensitive Measure Type and Valid to Change Condition CYBOCS-PDDClinician Yes Yes Resistance item (interview) Not relevant ABCStereotypy informant Yes Yes Only 7 items Repetitive informant Yesw (atNot ? Subscales vs. Behav Scale least one shown total score study)Stereotyped Behav informant Yes Not Fits for lower Scale (adults) shownfunctioning Repetitive Behav informant Yes Not Atyical scoringQuestionnaire shown

Anxiety can be assessed using one or more measures including, Anxiety,Depression and Mood Scale (ADAMS), Child and Adolescent SymptomInventory (CASI), Child Behavior Checklist (CBCL), MultidimensionalAnxiety Scale for Children (MASC), Pediatric Autism Rating Scale (PARS),Revised Child Anxiety and Depression Scale (RCAD), Screen for ChildAnxiety Related Disorders (SCARED). Nisonger Child Behavior Rating Form,and Anxiety Diagnostic Interview Scale (ADIS). Reliability and relevanceof some of these tools is presented below in Table 2.

TABLE 2 Clinical Tools Considered “Appropriate with Conditions” forEvaluating Anxiety Reliaable Sensitivity Measure Type & Valid to ChangeCondition CASI-Anxiety informant Yes Yes (pilot Incomplete data in ASD)coverage Multidimensional Informant Yes Yes (limited ⬆ reliance AnxietyScale for & Self- Use in ASD on language Children (MASC) report Pedi.Anxiety Clinician Yes Yes (limited ⬆ ⬆ reliance Rating Scale (interview)Use in ASD) on language (PARS) Anxiety Clinician Yes Yes (limited high-Diagnostic (interview) Use in ASD) functioning Interview Scaleindividuals

Potentially appropriate clinical tools for evaluating anxiety in ASDsand NDDs are shown below in Table 3.

TABLE 3 “Potentially Appropriate” Clinical Tools for Evaluating Anxietyin ASDs and NDDs Reliable Sensitive Measure Type & Valid To ChangeComment SCARED Informant Yes Yes (limited use ⬆ reliance on &self-report in ASD) language ADAMS informant Yes Not shonw Mood &anxiety Data in adults RCADS Informant Yes Not shown Mood & anxiety &self-report

Social communication can be assessed using clinical tools, for example,ABAS-II Domain scores, Aberrant Behavior Checklist (ABC)—Lethargy/SocialWithdrawal, ADI-R, Autism Diagnostic Observation Scale-Generic(ADOS-G)—new severity scores, Autism Impact Measure, Autism SpectrumRating Scales, Autism Treatment Evaluation Checklist (ATEC), Ball TossGame, Behavior Assessment Scale (BAS), Behavior Assessment System forChildren 2nd Edition BASC-2 (subscales relevant to social), BehaviorRating Inventory of Executive Function, California Verbal LearningTask-Children's Version (VLT-C) and Modified VLT-C (MVLT-C),Caregiver-Child Interaction, Jahromi 2009, CGI, Childhood Autism RatingScale (CARS), Children's Social Behavior Questionnaire, ClinicalEvaluation of Language Fundamentals (CELF-3 and 4)—Pragmatics Profile,Communication and Symbolic Behavior Scales (CSBS), Comprehension ofAffective Speech Task, General Trust Scale, Gilliam Autism Rating Scale(GARS), Joint Attention Measure from the ESCS (JAMES), Let's Face It!,Observational Assessment of Spontaneous Expressive Language (OSEL),Parent Questionnaire, Nagaraj et al. 2006, Parent's RatingQuestionnaire, Chan et al, 2009, Pervasive Developmental DisorderBehavior Inventory (PDD-BI) (Short version available: PDD-B 1-ScreeningVersion), Reading the Mind in Films-Adult, Reading the Mind inFilms-Child, Reading the Mind in the Eyes Task-Revised (RMET-R)-Adult,Reading the Mind in the Eyes Task-Revised (RMET-R)-Child, Reading theMind in Voice-Adult, Social Communication Questionnaire (SCQ), SocialResponsiveness Scale, Social Skills Improvement System (SSiS), Theory ofMind Test, and VABS-Socialization and Communication.

Of the tools used to assess social communication, the following areconsidered to be “Appropriate with Conditions:” Aberrant BehaviorChecklist (ABC): Lethargy/Social Withdrawal subscale, BASC-2: socialskills, withdrawal, functional subscale, CSBS, ESCS, JAMES, SSiS,Vineland Adaptive Behavior Scales socialization and communicationsubscales. Tools considered to be “Potentially Appropriate” includeABAS-II: conceptual and social domains, ADGS severity scores. AutismSpectrum rating Scales: social communication, CSBQ: understandingsubscales, and PDD-BI.

Autism

Classical Autism is a highly variable neurodevelopmental disorder. It istypically diagnosed during infancy or early childhood, with overtsymptoms often apparent from the age of 6 months, and becomingestablished by 2-3 years. According to the criteria set out in the DSM-5diagnosis of Autism requires a triad of symptoms to be present,including (a) impairments in social interaction, (b) impairments incommunication and (c) restricted and repetitive interests andbehaviours. Other dysfunctions, such as atypical eating, are also commonbut are not essential for diagnosis. Of these impairments, socialinteraction impairments are particularly important for diagnosis, andtwo of the following impairments must be present for a diagnosis ofautism:

-   -   (i) impairments in the use of multiple nonverbal behaviors        (e.g., eye contact) to regulate social interaction;    -   (ii) failure to develop peer relationships appropriate to        developmental level;    -   (iii) lack of spontaneous seeking to share enjoyment, interests,        or achievements;    -   (iv) lack of social or emotional reciprocity.

Communication impairments in Autism may be manifested in one or more ofthe following ways: delay in (or total lack of) the development ofspoken language; marked impairment in the ability to initiate or sustaina conversation; stereotyped and repetitive use of language; and/or alack of spontaneous make-believe play. Restricted, repetitive, andstereotyped patterns of behavior is also required for diagnosis, such aspreoccupation with one or more interest considered abnormal inintensity, inflexible adherence to routines or rituals, repetitive motormannerisms and/or persistent focus on parts of objects.

Lastly, for a diagnosis of Autism, it is necessary that the impairmentin the functioning of at least one area (i.e. social interaction,language, or imaginative play) should have an onset at less than 3 yearsof age.

Asperger Syndrome

Asperger Syndrome is similar to Autism and shares certain features. LikeAutism, Asperger Syndrome is also characterized by impairment in socialinteraction, and this is accompanied by restricted and repetitiveinterests and behavior. Thus, diagnosis of Asperger Syndrome ischaracterized by the same triad of impairments as Autism. However, itdiffers from the other ASDs by having no general delay in language orcognitive development and no deficit in interest in the subject'senvironment. Moreover, Asperger Syndrome is typically less severe insymptomology than classical Autism and Asperger's patients may functionwith self-sufficiency and lead relatively normal lives.

Childhood Disintegrative Disorder

Childhood disintegrative disorder (CDD), also known as Heller syndrome,is a condition in which children develop normally until age 2-4 years(i.e. later than in Autism and Rett Syndrome), but then demonstrate asevere loss of social, communication and other skills.

Childhood Disintegrative Disorder is very much like Autism, and bothinvolve normal development followed by significant loss of language,social play and motor skills. However, Childhood Disintegrative Disordertypically occurs later than Autism, involves a more dramatic loss ofskills, and is far less common.

Diagnosis of CDD is dependent on dramatic loss of previously acquiredskills in two or more of the following areas: language, social skills,play, motor skills (such as a dramatic decline in the ability to walk,climb, grasp, etc.), bowel or bladder control (despite previously beingtoilet-trained). The loss of developmental skills may be abrupt and takeplace over the course of days to weeks or may be more gradual.

Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS)

Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS) is anASD that describes patients exhibiting some, but not all, of thesymptoms associated with other well defined ASDs. The key criteria fordiagnosis of an ASD include difficulty socializing with others,repetitive behaviors, and heightened sensitivities to certain stimuli.These may all be found in the ASDs described above. However, Autism,Asperger Syndrome, Rett Syndrome and CDD all have other features thatenable their specific diagnosis. When specific diagnosis of one of thesefour disorders cannot be made, but ASD is apparent, a diagnosis ofPDD-NOS is made. Such a diagnosis may result from symptoms starting at alater age than is applicable for other conditions in the spectrum.

Rett Syndrome

Rett Syndrome (RTT) is a neurodevelopmental disorder that almostexclusively affects females (1 in 10:000 live births). Until recently,RTT was classified as an autism spectrum disorder (Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition-Revised(DSM-IV-R). Approximately 16,000 patients are currently affected by itin the U.S.A. (Rett Syndrome Research Trust data). For a diagnosis ofRett syndrome, the following symptoms are characteristic: impaireddevelopment from age 6-18 months; slowing of the rate of head growthstarting from between age 3 months and 4 years; severely impairedlanguage; repetitive and stereotypic hand movements; and gaitabnormalities, e.g. toe-walking or unsteady stiff-legged walk. There arein addition, a number of supportive criteria that may help diagnosis ofRett syndrome, but are not essential for a diagnosis. These includebreathing difficulties, EEG abnormalities, seizures, muscle rigidity andspasticity, scoliosis (curving of the spine), teeth-grinding, smallhands and feet in relation to height, growth retardation, decreased bodyfat and muscle mass, abnormal sleep patterns, irritability or agitation,chewing and/or swallowing difficulties, poor circulation andconstipation.

The onset of RTT usually begins between 6-18 months of age with aslowing of development and growth rates. This is followed by aregression phase (typically in children aged 1-4 years of age),pseudo-stationary phase (2-10 years of age) and a subsequent progressivelate motor deterioration state. RTT symptoms include sudden decelerationof growth and regression in language and motor skills includingpurposeful hand movements being replaced by stereotypical movements,autistic features, panic-like attacks, sleep cycle disturbances,tremors, seizures, respiratory dysfunctions (episodic apnea, hyperpnea),apraxia, dystonia, dyskinesia, hypotonia, progressive kyphosis orscoliosis and severe cognitive impairment. Most RTT patients surviveinto adulthood with severe disabilities and require 24-hour-a-day care.

Between 85% and 95% cases of RTT are reported to be caused by a mutationof the Mecp2 gene (Amir et al. 1999. Nat Genet 23:185-188; Rett SyndromeResearch Trust)—a gene encoding methyl-CpG-binding protein 2 (MeCP2).Mecp2 maps to the X-chromosome (location Xq28) and for this reason,mutations to the gene in males are usually lethal. While RTT is agenetic disorder, less than 1% of recorded cases are inherited; almostall mutations of Mecp2 occur de novo, with two thirds caused bymutations at 8 CpG dinucleotides (R106, R133, T158, R168, R255, R270,R294 and R306) located on the third and fourth exons.

MeCP2 is a protein that binds methylated CpG dinucleotides to exerttranscriptional silencing of DNA in the CNS. The key effect of areduction or absence of MeCP2 appears to be an impairment of dendriticspine development and the formation of synapses. MeCP2 expressionappears to temporally correlate with brain maturation, explaining whysymptoms typically appear around 18 months of age.

Presenting Features Common to ASDs

Taking the ASDs together, it is clear that there are commonalities inpresenting symptoms among all 5 forms. These common features areimpairments in normal social competences, and repetitive behaviours. Inall but Asperger Syndrome there is also a consistent presentation ofdelayed intellectual development most commonly manifest as a shortfallin language skills. Cognitive loss relative to normal parameters for theage is often quite marked in autism, Rett Syndrome, CDD and PDD-NOS.

Genetic Models of ASDs

To offer validity, animal models of ASDs must demonstrate similarsymptoms to the clinical conditions and have a reasonable degree of facevalidity regarding the etiology of those symptoms. It is known thatclassical Autism may be caused by many different genetic impairments andno single genetic defect is thought to account for more than a fewpercent of autism cases. Indeed, recent studies have revealed numerousde novo structural variations of chromosome locations thought tounderlie ASD, in addition to rare inherited genetic defects (Marshall etal, 2008; Sebat et al, 2007). Thus, copy number variation (CNV),translocation and inversion of gene sequences at 20 key sites or more,including 1p, 5q, 7q, 15q, 16p, 17p and Xq, have been mapped as ASDloci.

However, despite the polygenetic background underlying ASD and thecomplexity of the etiology, it is known that certain genetic defects canproduce ASD. Some of the best characterized defects arise fromchromosomal aberrations of genes that code for a cluster of postsynapticdensity proteins, including neuroligin-3 (NLGN3), neuroligin-4 (NLGN4),neurexin-la (NRXN1) and shank3 (Sebat et al, 2007).

NLGN3 and NLGN4 are postsynaptic cell-adhesion molecules present inglutamatergic synapses. They play a role of coordinating presynapticcontact to the postsynaptic site and also interact with the postsynapticscaffolding protein shank3. Mutations to NLGN3 and NLGN4 have beenobserved in the ASD population and account for perhaps 1% of all ASDcases (Lintas & Persico, 2008). Jamain and colleagues first reported amissense to NLGN3 and a frameshift to NLGN4 in two unrelated subjects,resulting in Asperger Syndrome and classical Autism, respectively(Jamain et al, 2003). While the incidence of NLGN3 or NLGN4 mutations inthe ASD population is certainly low (indeed, no such mutations wereobserved in a study of 96 ASD patients in a Canadian study; Gauthier etal, 2003), it has been confirmed in preclinical studies that neuroliginmutations can indeed produce of model of autistic symptoms. Thus,introduction to mice of the same R431C missense to NLGN3 that has beenreported clinically results in a mutant mouse strain showing reducedsocial interaction and enhanced inhibitory synaptic transmission(Tabuchi et al, 2007).

The R431C mutant therefore mouse represents a model for ASD based uponNLGN3 mutation. In this case, mutation at the R451 position of NLGN3results in a ‘gain-of-function’ mutation.

In contrast, modeling the clinical mutation of NLGN4 in mice is achievedby a ‘loss-of-function’ mutation of NLGN4 (a classical knockout model).In this model, mutant mice display a social interaction deficit andreduced ultrasonic vocalization (Jamain et al, 2008). Communicationdeficits are central to clinical ASDs and in the NLGN4 knockout mice areduction in ultrasonic vocalizations from male mice exposed towild-type female counterparts supports the face validity of the strainas a model of ASD.

Presynaptic neurexin proteins induce postsynaptic differentiation inapposing dendrites through interactions with postsynaptic neuroligincounterparts. Mutations of the neurexin-la (NRXN1) gene have beenreported in numerous studies (Sebat et al, 2007; Marshall et al, 2008;Kim et al, 2008; Yan et al, 2008) and these have been observed in theform of copy-number variants. As with NLGN mutations, when a mutation ofthe NRXN1 gene is introduced to mice (in the form of gene knockout), amutant strain with certain ASD-like features is produced (Etherton etal, 2009). These NRXN1 knockout mice show a decrease in hippocampalminiature excitatory postsynaptic current (mEPSC) frequency and adecreased input-output relationship of evoked currents. Theseelectrophysiological effects relate to decreased excitatory transmissionin the hippocampus. In addition to decreased excitatoryneurotransmission, NRXN1 knockout mice exhibit a decrease in pre-pulseinhibition, though social behaviour appears to be unaffected (Ethertonet al, 2009).

Sharing certain features with the neurexin-NLGN trans-synapticconstruct, cell adhesion molecule 1 (CADM1) is an immunogolbulin familyprotein present both pre- and post-synaptically that is also involved insynaptic trans-cell adhesion activity (Biederer et al, 2002). Mutationsto the CADM1 gene have been detected in ASD patients and appear torepresent a further possible cause of these conditions (Zhiling et al,2008).

Analysis of CADM1 knockout mice reveals that these animals showincreased anxiety-related behavior, impaired in social interaction andimpaired social memory and recognition. In addition CADM1 knockout micedemonstrate poorer motor skills (Takayanagi et al, 2010). Thesedysfunctions are again consistent with ASD symptomology.

22q13 deletion syndrome (also known as Phelan-McDermid Syndrome), is arare genetic disorder caused by a microdeletion at the q13.3 terminalend of chromosome 22. This microdeletion is rarely uncovered by typicalgenetic screening and a fluorescence in situ hybridization test isrecommended to confirm the diagnosis. Recent work indicates the syndromeis caused by errors in the gene shank3 which codes for a postsynapticdensity protein critical for normal neuronal functioning. Interestingly,errors in this gene have also been associated with ASD and 22q13deletion syndrome can commonly lead to an ASD diagnosis (Durand et al,2007; Moessner et al, 2007; Sykes et al, 2009). Given the closeassociation of 22q13 deletion syndrome and the consequential diagnosisof ASD, a mutant mouse model of this mutation has been developed.

The shank3 knockout mouse exhibits several deficits that mirror ASDsymptoms, including reduced ultrasonic vocalizations (i.e., diminishedsocial communication) as well as impaired social interaction timebetween mice. In addition, these mice also have impaired hippocampal CA1excitatory transmission, measured by input-output relationship of evokedcurrents and impaired long-term potentiation (LTP). LTP is believed tobe a physiological process underlying memory formation andconsolidation. Thus, the model exhibits a similar phenotype to the NLGN4knockout, consistent with ASD.

As has been noted, 22q13 deletion syndrome itself is very rare. However,it provides important information that involvement of specific genes mayhave a definitive role in the etiology of ASDs. In addition to shank3,this disorder reveals a further possible gene defect in ASD. Of the 50or so cases of 22q13 deletion syndrome described, all but one have agene deletion that extends beyond shank3 to include a further gene,known as the Islet Brain-2 gene (IB2) (Sebat et al, 2007). The IB2protein interacts with many other proteins including MAP kinases andamyloid precursor protein, appears to influence protein trafficking inneurites and is enriched at postsynaptic densities (Giza et al, 2010).Mice lacking the protein (IB2−/− knockout mice) exhibit impaired socialinteraction (reduced social sniffing and interaction time), reducedexploration and cognitive and motoric deficits (Giza et al, 2010). Thisbehavioural phenotype was associated with reduced excitatorytransmission in cerebellar cells. As with shank3 knockout, the phenotypeof IB2 mutation is therefore also consistent with ASD.

In addition to the animal models of postsynaptic density protein defectsdescribed above, other monogenetic syndromes that share various featureswith ASDs can lead to autism offer another avenue for drug targeting ofASD.

Recently, Fragile X Syndrome has been assigned to another family ofdisorders, called Neurodevelopmental Disorders (NDDs). The descriptionsherein make no distinction based on the official classification of thedisorder. If, in the future, one or other ASD or NDD is reclassified,this descriptions and disclosures herein will apply to those newclassifications, regardless of their name(s).

Fragile X Syndrome

Fragile X Syndrome (FXS) is caused by the expansion of a singletrinucleotide gene sequence (CGG) on the X-chromosome that results infailure to express the protein coded by the fmr1 gene. FMR1 (fragile Xmental retardation 1) is a protein required for normal neuraldevelopment. Fragile X Syndrome can cause a child to have autism(Hagerman et al, 2010); in 2-6% of all children diagnosed with Autismthe cause is FMR1 gene mutation. Moreover, approximately 30% of childrenwith FXS have some degree of Autism and a further 30% are diagnosed withPDD-NOS (Hagerman et al, 2010). Indeed, Fragile X Syndrome is the mostcommon known single gene cause of Autism. FMR1 knockout mice have beendeveloped as a model of FXS, and therefore, as a further model amutation of the fmr1 gene has been shown to result in abnormal dendriticspine development and pruning (Comery et al, 1997), along with anassociated dysregulation of dendritic scaffold proteins (includingshank 1) and glutamate receptor subunits in postsynaptic densities(Schütt et al, 2009). These effects of dendrite morphology result inimpaired LTP in the cortex and amygdala (Zhao et al, 2005) andhippocampus (Lauterbom et al, 2007), as well as impaired cognition(Kreuger et al, 2011) and an enhancement in social anxiety (Spencer etal, 2005).

Rett Syndrome

In contrast to the ASD of autism, Asperger, CDD and PDD-NOS, RettSyndrome appears to have an almost monogenetic basis and may be modelledin mice with good face validity. Rett Syndrome is thought be caused, inup to 96% of cases, by a defect in the Mecp2 gene (Zoghbi, 2005). As aresult, MeCP2 knockout mutant mice provide an animal model with all thehallmarks of clinical Rett Syndrome, with a phenotype showing someoverlap with the NLGN4, shank3 and IB2 knockout models of ASD. Thus,MeCP2 knockout mice display a clear impairment in LTP in the hippocampusalong with a corresponding decrease in social and spatial memory(Moretti et al, 2006) and impaired object recognition (Schaevitz et al,2010).

Thus, ASDs in human beings share many features of cognitive ordevelopmental disorders in animals, including rodents. Therefore,studies of therapies of ASDs in rodents such as mice and rats arereasonably predictable of results obtained in human beings.

Compounds of the Invention

Certain embodiments of this invention include novel derivatives ofcyclic Prolyl-Glutamate (“cPG”) having structures as described below.

In certain embodiments, compounds of Formula 1 include substituentswhere:

X¹ is selected from the group consisting of NR′, O and S;

X² is selected from the group consisting of CH₂, NR′, O and S;

R¹, R², R³, R⁴ and R⁵ are independently selected from the groupconsisting of —H, —OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′,—C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl, halogen, alkyl, substitutedalkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl,heteroarylalkyl and substituted heteroarylalkyl; each R′ isindependently selected from the group consisting of —H, alkyl,heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl;

-   -   or R⁴ and R⁵ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6;    -   or R² and R³ taken together are —CH₂—(CH₁)_(n)—CH₂— where n is        an integer from 0-6;        with the proviso that when R¹=methyl and R²=R³=R⁴=H then        R⁵≠benzyl and;        when R¹=H, at least one of R² and R³≠H.

In further embodiments, compounds of Formula 1 include substituentswhere:

-   -   R¹=methyl, R²=R³=R⁴=R⁵=H, X¹=NH, X²=CH₂;    -   R¹=allyl, R²=R³=R⁴=R⁵=H, X¹=NH, X²=CH₂;    -   R¹=R⁴=R⁵=H, R²=R³=methyl, X¹=NH, X²=CH₂;    -   R¹=R⁴=R³=H, R²=R³=methyl, X¹=NH, X²=CH₂.

In other embodiments of the invention, compounds of Formula 1 includesubstituents where;

-   -   R⁴ and R⁵ taken together are —CH₂—(CH₂)_(n)—CH₂— and:    -   R¹=methyl, R²=R³=H, n=0, X¹=NH, X²=CH₂;    -   R¹-methyl, R²=R³=H, n=2, X¹=NH, X²=CH₂;    -   R¹=allyl, R²=R³=H, n=0, X¹=NH, X²=CH₂;    -   R¹=allyl, R²=R³=H, n=2, X¹=NH, X²=CH₂.    -   R¹-methyl, R²=R³=H, n=3, X¹=NH, X²=CH₂;    -   R¹=allyl, R²=R³=H, n=3, X¹=NH, X²=CH₂.

In still other embodiments of the invention, compounds of Formula 1include substituents where R¹=methyl or allyl, R²=R³=R⁴=H and R⁵ isselected from the group consisting of the side chains of the aminoacids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, histidine, isoleucine, leucine, lysine, methionine,proline, serine, threonine, tryptophan, tyrosine, valine, norvaline,norleucine, citruline, ornithine, homocysteine, homoserine,alloisoleucine, isovaline, sarcosine and the like.

In yet further embodiments of the invention, compounds of Formula 1include substituents where:

-   -   R¹=methyl, R²=R³=methyl, R⁴=R⁵=H, X¹=NH and X²S;    -   R¹-allyl, R²=R³=methyl, R⁴=R⁵H, X¹=NH, and X²=S.

Those with skill in the art will appreciate that the above structuralrepresentations can contain chiral centres, the number of which willdepend on the different substituents. The chirality may be either R or Sat each centre. The structural drawings can represent only one of thepassible tautomeric, conformational diastereomeric or enantiomericforms, and it should be understood that the invention encompasses anytautomeric, conformational isomeric diastereomeric or enantiomeric form,which exhibits biological or pharmacological activity as describedherein.

Pharmacology and Utility

Cyclic Glycyl-2-Allyl Proline (cG-2-AllylP) is described in U.S. Utilityapplication Ser. No. 11/399,974 filed Apr. 7, 2006, entitled “CyclicG-2Allyl Proline in Treatment of Parkinson's Disease,” now U.S. Pat. No.7,776,876, issued Aug. 17, 2010, U.S. Utility application Ser. No.10/570,393, filed Mar. 2, 2006 entitled “Neuroprotective BicyclicCompounds and Methods for Their Use,” now U.S. Pat. No. 8,067,425, PCTInternational Patent Application No: PCT/US2004/028308, entitled“Neuroprotective Bicyclic Compounds and Methods for Their Use,” U.S.Provisional Patent Application Ser. No. 60/499,956 filed Sep. 3, 2003,entitled “Neuroprotective Bicyclic Compounds and Methods for Their Use,”and U.S. patent application Ser. No. 13/043,215 filed Mar. 8, 2011,entitled “Cyclic Glycyl-2-AllylProline Improves Cognitive Performance inImpaired Animals.” Each of the above patent applications and patents isexpressly incorporated herein fully by reference.

Certain aspects of this invention include the use of cyclic G-2-AllylPin treatment of cognitive impairment associated with aging withneurodegenerative conditions or in situations in which cognitiveimpairment is found with no apparent neurodegeneration.

Scopolamine is commonly used in animal models of cholinergichypofunction associated with Alzheimer's disease. The functionaldeficits observed after scopolamine treatment include those found inhuman patients with Alzheimer's disease. Thus, scopolamine treatment isreasonably predictive of cognitive impairment found in human diseases.Additionally, scopolamine treatment mimics cognitive dysfunction inhumans who do not have neurodegenerative disorders.

cG-2-AllylP administered to animals treated with scopolamine-inducedcognitive dysfunction produces clinical improvement in those animals,similar to the therapeutic improvement observed in people suffering fromcholinergic hypofunction. For example, cholinergic hypofunctionassociated with Alzheimer's disease. Thus, studies of effects of CyclicG-2-AllylP scopolamine treated animals are reasonably predictive ofeffects observed in human beings suffering from cholinergic dysfunction.

Other agents can be administered along with a compound of thisinvention. Such other agents may be selected from the group consistingof for example, growth factors and associated derivatives, e.g.,insulin-like growth factor-1 (IGF-I), insulin-like growth factor-II(IGF-U), the tripeptide GPE, transforming growth factor-β1, activin,growth hormone, nerve growth factor, growth hormone binding protein,and/or IGF-binding proteins. Additional compounds includeGlycyl-2-Methyl Prolyl Glutamate and/or other compounds disclosed inU.S. patent application Ser. No. 10/133,864, now U.S. Pat. No.7,041,314, issued May 9, 2006, expressly incorporated herein fully byreference.

Therapeutic Applications

Compositions and methods of the invention find use in the treatment ofanimals, such as human patients, suffering from cognitive impairment andsymptoms associated with ASDs and NDDs. Still more generally, thecompositions and methods of the invention find use in the treatment ofmammals, such as human patients, suffering from memory impairment,intellectual disability, impaired social interaction, impairments incommunication, restricted and repetitive interests and behaviours andseizures.

Pharmaceutical Compositions and Administration

Cyclic G-2-AllylP can be administered as part of a medicament orpharmaceutical preparation. This can involve combining a compound of theinvention with any pharmaceutically appropriate carrier, adjuvant orexcipient. The selection of the carrier, adjuvant or excipient will ofcourse usually be dependent upon the route of administration to beemployed.

In general, compounds of this invention will be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with other conventional therapeuticagents for the disease being treated. A therapeutically effective amountmay vary widely depending on the disease or injury, its severity, theage and relative health of the animal being treated, the potency of thecompound(s), and other factors. As anti-apoptotic, anti-inflammatory andanti-necrotic agents, therapeutically effective amounts of cyclicG-2-AllylP may range from 0.001 to 100 milligrams per kilogram mass ofthe animal, with lower doses such as 0.001 to 0.1 mg/kg beingappropriate for administration through the cerebrospinal fluid, such asby intracerebroventricular administration, and higher doses such as 1 to100 mg/kg being appropriate for administration by methods such as oral,systemic (e.g. transdermal), or parenteral (e.g. intravenous)administration. A person of ordinary skill in the art will be ablewithout undue experimentation, having regard to that skill and thisdisclosure, to determine a therapeutically effective amount of acompound of this invention for a given disease or injury.

Cyclic G-2-AllylP and other cGP related compounds may be administeredperipherally via any peripheral route known in the art. These caninclude parenteral routes for example injection into the peripheralcirculation, subcutaneous, intraorbital, ophthalmic, intraspinal,intracisternal, topical, infusion (using e.g. slow release devices orminipumps such as osmotic pumps or skin patches), implant, aerosol,inhalation, scarification, intraperitoneal, intracapsular,intramuscular, intranasal, oral, buccal, transdermal, pulmonary, rectalor vaginal. The compositions can be formulated for parenteraladministration to humans or other mammals in therapeutically effectiveamounts (e.g. amounts which eliminate or reduce the patient'spathological condition) to provide therapy for the neurological diseasesdescribed above.

Desirably, if possible, when administered as anti-apoptotic,anti-inflammatory and anti-necrotic agent, cyclic G-2-AllylP can beadministered orally. The amount of a compound of this invention in thecomposition may vary widely depending on the type of composition, sizeof a unit dosage, kind of excipients, and other factors well known tothose of ordinary skill in the art. In general, the final compositionmay comprise from 0.0001 percent by weight (% w) to 10% w of thecompound of this invention, preferably 0.001% w to 1% w, with theremainder being the excipient or excipients.

Other convenient administration routes include subcutaneous injection(e.g. dissolved in a physiologically compatible carrier such as 0.9%sodium chloride) or direct administration to the CNS. Using stereotacticdevices and accurate maps of an animals' CNS, a compound may be injecteddirectly into a site of neural damage. Such routes of administration maybe especially desired in situations in which perfusion of that locationis compromised either by decreased vascular perfusion or by decreasedcerebral spinal fluid (CSF) flow to that area. Examples includeadministration by lateral cerebroventricular injection or through asurgically inserted shunt into the lateral cerebroventricle of the brainof the patient, intravenously, direct injection into the desiredlocation, either directly or indirectly via the circulation, or otherroutes.

By “directly or indirectly via the circulation” we mean administrationof cG-2-AllylP to any tissue that has blood flow sufficient to deliverthe agent into the circulation. Non-limiting examples include the skin,nose, pharynx, gastrointestinal tract, or other such tissue. Whenadministered to such a tissue, the agent is absorbed by the tissue,where the agent enters the interstitial fluid of the tissue, andsubsequently is absorbed by venules, capillaries, arterioles or lymphducts. The agent is then carried into the general systemic circulation,where it can be delivered to the affected site, including the brain.When the agent is administered subcutaneously or peritoneally, the agentis absorbed by an adjacent tissue, and the agent then enters thecirculation locally, and subsequently is delivered to the generalcirculation, where it can be transported to the brain. When the agentapproaches the blood-brain barrier, the agent then can diffuse into thebrain, either to neural tissue, or into the cerebrospinal fluid, whereit can be delivered to neural tissues.

The effective amount of compound in the CNS may be increased byadministration of a pro-drug form of a compound, which comprises acompound of the invention and a carrier, where the carrier is joined toa compound of the invention by a linkage which is susceptible tocleavage or digestion within the patient. Any suitable linkage can beemployed which will be cleaved or digested following administration.

However, there is no intention on the part of the applicants to excludeother forms of administration.

In further embodiments of the invention, restoring nerve function in ananimal can comprise administering a therapeutic amount of cyclicG-2-AllylP in combination with another neuroprotective agent, selectedfrom, for example, growth factors and associated derivatives(insulin-like growth factor-I (IGF-I), insulin-like growth factor-II(IGF-II), transforming growth factor-01, activin, growth hormone, nervegrowth factor, growth hormone binding protein, IGF-binding proteins(especially IGFBP-3), basic fibroblast growth factor, acidic fibroblastgrowth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6,keratinocyte growth factor, androgen-induced growth factor. Additionalmembers of the FGF family include, for example, int-2, fibroblast growthfactor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, karatinocytegrowth factor 2, glial-activating factor, FGF-10 and FGF-16, ciliaryneurotrophic factor, brain derived growth factor, neurotrophin 3,neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial-cell linederived neurotrophic factor, activity-dependent neurotrophic factor,cytokine leukaemia inhibiting factor, oncostatin M, interleukin), α-,β-, γ-, or consensus interferon, and TNF-α. Other forms ofneuroprotective therapeutic agents include, for example, clomethiazole;kynurenic acid, Semax, tacrolimus,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,adrenocorticotropin-(4-9) analogue (ORG 2766) and dizolcipine (MK-801),selegiline; glutamate antagonists such as, NPS 1506, GV1505260, MK-801,GV150526; AMPA antagonists such as2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070and LY300164; anti-inflammatory agents directed against the addressinMAdCAM-1 and/or its integrin α4 receptors (α4β1 and α4β7), such asanti-MAdCAM-1 mAb MECA-367 (ATCC accession no. HB-9478).

Cyclic G-2-AllylP and other cGP related compounds are suitablyadministered by a sustained-release system. Suitable examples ofsustained-release compositions include semi-permeable polymer matricesin the form of shaped articles, e.g., films, or microcapsules.Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919; EP 38,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers: 22: 547-36),poly(2-hydroxyethyl methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res.: 15: 267), ethylene vinyl acetate (Langer et al., 1981, J.Biomed. Mater. Res.: 15: 267), or poly-D-(−)-3-hydroxybutyric acid (EP133,988). Sustained-release compositions also include a liposomallyentrapped compound. Liposomes containing the compound are prepared bymethods known per se: DE 3,218,121, EP 52,322, EP 36,676, EP 88,046, EP143,949, EP 142,641, Japanese Pat. Appln. 83-118008, U.S. Pat. Nos.4,485,045 and 4,544,545, and EP 102,324. Ordinarily, the liposomes areof the small (from or about 200 to 800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol percentcholesterol, the selected proportion being adjusted for the mostefficacious therapy.

For parenteral administration, in one embodiment cyclic G-2-AllylP canbe formulated generally by mixing each at the desired degree of purity,in a unit dosage injectable form (solution, suspension, or emulsion),with a pharmaceutically, or parenterally, acceptable carrier, i.e., onethat is non-toxic to recipients at the dosages and concentrationsemployed and is compatible with other ingredients of the formulation.

For delivery of a compound of this invention to a mucosal tissue, onecan incorporate the compound into a gel formulation. Once delivered tothe mucosa (e.g., oral cavity, gastrointestinal tract, rectum), theagent can diffuse out of the gel, or the gel can be degraded, therebyreleasing the agent into the tissue, where it can be absorbed into thecirculation. Exemplary gel formulations can include those made withcarboxypolysaccharides such as carboxymethyl cellulose, carboxyethylcellulose, chitin, chitosan, starch, cellulose, proteins such ashyaluronic acid, or other polymers, such as polyvinylpyrollidine,polyvinyl alcohols, as well as other gel materials known in the art

Generally, the formulations are prepared by contacting cyclic G-2-AllylPwith liquid carriers or finely divided solid carriers or both. Then, ifnecessary, the product is shaped into the desired formulation.Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, abuffered solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein.

A carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

Cyclic G-2-AllylP and other cGP compounds typically formulated in suchvehicles at a pH of from or about 4.5 to 8. It will be understood thatuse of certain of the foregoing excipients, carriers, or stabilizerswill result in the formation of salts of the compound. The finalpreparation may be a stable liquid or lyophilized solid.

Formulations of cyclic G-2-AllylP in pharmaceutical compositions canalso include adjuvants. Typical adjuvants which may be incorporated intotablets, capsules, and the like are a binder such as acacia, cornstarch, or gelatin; an excipient such as microcrystalline cellulose; adisintegrating agent like corn starch or alginic acid; a lubricant suchas magnesium stearate; a sweetening agent such as sucrose or lactose; aflavouring agent such as peppermint, wintergreen, or cherry. When dosageforms are tablets, cyclic G-2-AllylP compositions can include bindersand optionally, a smooth coating. When the dosage form is a capsule, inaddition to the above materials, it may also contain a liquid carriersuch as a fatty oil. Other materials of various types may be used ascoatings or as modifiers of the physical form of the dosage unit. Asyrup or elixir may contain the active compound, a sweetener such assucrose, preservatives like propyl paraben, a colouring agent, and aflavouring agent such as cherry. Sterile compositions for injection canbe formulated according to conventional pharmaceutical practice. Forexample, dissolution or suspension of the active compound in a vehiclesuch as water or naturally occurring vegetable oil like sesame, peanut,or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired, buffers, preservatives, antioxidants, and the likecan be incorporated according to accepted pharmaceutical practice.

For injection, intraventricular administration, and other invasiveroutes of administration, cyclic G-2-AllylP must be sterile. Sterilitymay be accomplished by any method known in the art, for examplefiltration through sterile filtration membranes (e.g., 0.2 micronmembranes). Therapeutic compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper able to be pierced by a hypodermicinjection needle.

A pharmaceutical formulation containing cyclic G-2-AllylP ordinarilywill be stored in unit or multi-dose containers, for example, in sealedampoules or vials, as an aqueous solution or as a lyophilizedformulation for reconstitution. As an example of a lyophilizedformulation, 10 mL vials are filled with 5 mL of sterile-filtered 1%(w/v) aqueous solution of compound, and the resulting mixture islyophilized. The infusion solution is prepared by reconstituting thelyophilized compound using bacteriostatic Water-for-Injection. It can bereadily appreciated that other dosage forms and types of preparationscan be used, and all are considered to be part of this invention.

Preparation of the Compounds

Starting materials and reagents used in preparing cyclic G-2-AllylP areeither available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to the person of ordinaryskill in the art following procedures described in such references asFieser and Fieser's Reagents for Organic Synthesis, vols 1-17, JohnWiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of CarbonCompounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989;Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y.,1991; March J; Advanced Organic Chemistry, 4^(th) ed. John Wiley andSons, New York, N.Y., 1992; and Larock: Comprehensive OrganicTransformations, VCH Publishers, 1989. In most instances, amino acidsand their esters or amides, and protected amino acids, are widelycommercially available; and the preparation of modified amino acids andtheir amides or esters are extensively described in the chemical andbiochemical literature and thus well-known to persons of ordinary skillin the art.

Starting materials, intermediates, and final products this invention maybe isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data.

Cyclic G-2-AllylP is a cyclic dipeptide (bicyclic 2,5-diketopiperazine),and is a member of the class of compounds known as cyclic GPx (“cGP”).In general, cGPs and cyclic G-2-AllylP may be prepared by methods suchas are already well-known to persons of ordinary skill in the art ofpeptide and modified peptide synthesis, following the reaction schemesset forth in the Figures following this specification, or by followingother methods well-known to those of ordinary skill in the art of thesynthesis of peptides and analogues. See for example, Bodanzsky:Principles of Peptide Synthesis, Berlin, N.Y.: Springer-Verlag 1993.

Synthesis of the diketopiperazine compounds of this invention may be bysolution-phase synthesis as discussed in the Examples or via thesolid-phase synthesis method exemplified by Merrifield et al. 1963 J.Amer. Chem. Soc.: 83, 2149-2136. Solid phase synthesis may be performedusing commercial peptide synthesizers, such as the Applied BiosystemsModel 430A, using the protocols established for the instrument.

Specific examples of diketopiperazine synthesis can be found in theExamples following and in, for example, Fischer, 2003, J. PeptideScience: 9:9-33 and references therein. A person of ordinary skill inthe art will have no difficulty, taking account of that skill and theknowledge available, and of this disclosure, in developing one or moresuitable synthetic methods for compounds of this invention.

The choice of appropriate protecting groups for the method chosen(solid-phase or solution-phase), and of appropriate substrates ifsolid-phase synthesis is used, will be within the skill of a person ofordinary skill in the art. Appropriate protecting groups for peptidesynthesis include r-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl(Fmoc), Benzyl (Bzl), r-amyloxycarbonyl (Aoc), tosyl (Tos),benzyloxycarbonyl (Z or Cbz), o-bromo-benzyloxycarbonyl (BrZ) and thelike. Additional protecting groups are identified in Goodman M. (ed.),“Synthesis of Peptides and Peptidomimetics” in Methods of organicchemistry (Houben-Weyl) (Workbench Edition, E22a,b,c,d,e; 2004; GeorgThieme Verlag, Stuttgart, N.Y.).

The choice of coupling agent for the method chosen will also be withinthe skill of a person of ordinary skill in the art. Suitable couplingagents include DCC (N, N′-Dicyclohexylcarbodiimide), Bop(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate), PyBop(Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate),BopCl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride),2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) and thelike. Other compounds may be used in the synthesis e.g. to preventracemisation, such as HOBt (N-Hydroxybenzotriazole) and HOAt(1-Hydroxy-7-azabenzotriazole).

EMBODIMENTS

The specific embodiments presented below are not intended to be limitingto the scope of the invention. Persons of skill in the art can createother embodiments by incorporating one or more of the elements in thelisting below into combinations not specifically set forth herein. Allsuch embodiments are considered to be within the scope of the invention.

Embodiment 1. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound having the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   X¹ is selected from the group consisting of NR′, O and S;    -   X² is selected from the group consisting of CH₂, NR′, O and S;    -   R¹, R², R³, R⁴ and R⁵ are independently selected from the group        consisting of —H, —OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′,        —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl, halogen,        alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        arylalkyl, substituted arylalkyl, heteroarylalkyl and        substituted heteroarylalkyl; each R′ is independently selected        from the group consisting of —H, alkyl, heteroalkyl, alkenyl,        alkynyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl;    -   or R⁴ and R⁵ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6;    -   or R² and R³ taken together are —CH₂(CH₂)_(n)—CH₂— where n is an        integer from 0-6;    -   with the proviso that when R¹=methyl and R²=R³=R⁴=H then        R⁵≠benzyl and;    -   when R¹=H, at least one of R² and R³≠H.

Embodiment 2. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound has the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   X¹ is selected from the group consisting of NR′, O and S;    -   X² is selected from the group consisting of CH₂, NR′, O and S;    -   R¹, R² and R³ are independently selected from the group        consisting of group consisting of —H, —OR′, —SR′. —NR′R′, —NO₂,        —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl,        halogen, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl        and substituted heteroarylalkyl; each R′ is independently        selected from the group consisting of —H, alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and        heteroarylalkyl;    -   or R² and R³ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6;    -   with the proviso that at least one R 4≠H.

Embodiment 3. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound has the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   X¹ is selected from the group consisting of NR′, O and S;    -   X² is selected from the group consisting of CH₂, NR′, O and S;    -   R¹, R² and R³ are independently selected from the group        consisting of group consisting of —H, —OR′, —SR′, —NR′R′, —NO₂,        —CN, —C(O)R′, —C(O)OR′. —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl,        halogen, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl        and substituted heteroarylalkyl; each R′ is independently        selected from the group consisting of —H, alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and        heteroarylalkyl;    -   or R² and R³ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6.

Embodiment 4. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   X¹, X³, and X⁴ are independently selected from the group        consisting of S, O, and NH;    -   X² is selected from the group consisting of S, O, CH₂ and NH;    -   R¹, R², R³, R⁴ and R³ are independently selected from the group        consisting of —H, —OR′, —SR′, —NR′R′, —NO₂. —CN, —C(O)R′,        —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl, halogen,        alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        arylalkyl, substituted arylalkyl, heteroarylalkyl and        substituted heteroarylalkyl; each R′ is independently selected        from the group consisting of —H, alkyl, heteroalkyl, alkenyl,        alkynyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl;    -   or R⁴ and R³ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6;    -   or R² and R³ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6;    -   with the proviso that at least one R≠H and that both X³ and        X⁴≠O.

Embodiment 5. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound of the formula;

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   R¹ and R² are independently selected from the group consisting        of group consisting of —H, —OR′, —SR′, —NR′R′, —NO₂, —CN,        —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl,        halogen, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl        and substituted heteroarylalkyl; each R′ is independently        selected from the group consisting of —H, alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and        heteroarylalkyl;    -   or R¹ and R² taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6.

Embodiment 6. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   R¹, R² and R¹ are independently selected from the group        consisting of group consisting of —H, —OR′, —SR′, —NR′R′, —NO₂,        —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl,        halogen, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl        and substituted heteroarylalkyl; each R′ is independently        selected from the group consisting of —H, alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and        heteroarylalkyl;    -   or R² and R³ taken together are —CH₂—(CH₂)_(n)—CH₂— where n is        an integer from 0-6.

Embodiment 7. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

-   -   R is selected from the group consisting of alkyl, substituted        alkyl, heteroalkyl, substituted heteroalkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl,        substituted arylalkyl, heteroarylalkyl and substituted        heteroarylalkyl.

Embodiment 8. The method of any of embodiments 1 to 4 or 6 whereR¹=methyl.

Embodiment 9. The method of any of embodiments 1 to 4 or 6 whereR¹=allyl.

Embodiment 10. The method of any of embodiments 1 to 4 whereR²=R³=methyl and X²=S.

Embodiment 11. The method of embodiment 1 where R¹=allyl, R²=R³=R⁴=R⁵=H,X¹=NH, X²=CH₂.

Embodiment 12. The method of embodiment 1 where R¹=methyl, R²=R³=H, R⁴and R⁵ taken together are —CH₂—(CH₂)₃—CH₂—, X¹=NH, X²=CH₂.

Embodiment 13. The method of embodiment 1 where R¹=methyl, R²=R³=H, R⁴and R⁵ taken together are —CH₂—(CH₂)₂—CH₂—, X¹=NH, X²=CH₂.

Embodiment 14. The method of any of embodiments 1 to 13, furthercomprising administering a pharmaceutically acceptable excipient.

Embodiment 13. The method of any of embodiments 1 to 13, furthercomprising administering a pharmaceutically acceptable excipient and abinder.

Embodiment 16. The method of any of embodiments 1 to 13, furthercomprising administering a pharmaceutically acceptable excipient and acapsule.

Embodiment 17. The method of any of embodiments 1 to 13, furthercomprising administering at least one other anti-apoptotic,anti-necrotic or neuroprotective agent.

Embodiment 18. The method of embodiment 17 where the otheranti-apoptotic or neuroprotective agent is selected from selected fromgrowth factors and associated derivatives (insulin-like growth factor-I[IGF-I], insulin-like growth factor-II [IGF-II], transforming growthfactor-β1, activin, growth hormone, nerve growth factor, growth hormonebinding protein, IGF-binding proteins [especially IGFBP-3], basicfibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgkgene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor,androgen-induced growth factor, int-2, fibroblast growth factorhomologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growthfactor 2, glial-activating factor, FGF-10 and FGF-16, ciliaryneurotrophic factor, brain derived growth factor, neurotrophin 3,neurotrophin 4, bone morphogenetic protein 2 [BMP-2], glial-cell linederived neurotrophic factor, activity-dependent neurotrophic factor,cytokine leukaemia inhibiting factor, oncostatin M, an interleukin,α-interferon, β-interferon, γ-interferon, consensus interferon, TNF-α,clomethiazole; kynurenic acid, Semax, tacrolimus,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,adrenocorticotropin-(4-9) analogue [ORG 2766], dizolcipine [MK-801],selegiline, a glutamate antagonist, an AMPA antagonist, and ananti-inflammatory agent.

Embodiment 19. The method of embodiment 18 wherein said glutamateantagonist is selected from the group consisting of NPS1506, GV1505260,MK-801, and GV150526.

Embodiment 20. The method of embodiment 18 wherein said AMPA antagonistis selected from the group consisting of2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070and LY300164.

Embodiment 21. The method of embodiment 18, wherein saidanti-inflammatory agent is selected from the group consisting of ananti-MAdCAM-1 antibody and an antibody against an integrin α4β1 receptorand an integrin α4β7 receptor.

Embodiment 22. The method of embodiment 21 wherein said anti-MAdCAM-1antibody is MECA-367.

Embodiment 23. The method of embodiment 1, wherein said compound iscyclic G-2-AllylP.

Embodiment 24. The method of embodiment 1, wherein said compound iscyclic cyclohexyl-G-2MeP.

Embodiment 23. The method of embodiment 1, wherein said compound iscyclic cyclopentyl-G-2MeP.

Embodiment 26. A method for treating a symptom of an Autism SpectrumDisorder (ASD) or Neurodevelopmental Disorder (NDD) in an animalsuffering from such a disorder, comprising administering to the animal,a pharmaceutically effective amount of cyclic Glycyl-2-Allyl Proline(cG-2-AllylP) to said mammal.

Embodiment 27. The method of embodiment 26, wherein said cG-2-AllylPcomprises an aqueous solution and one or more pharmaceuticallyacceptable excipients, additives, carriers or adjuvants.

Embodiment 28. The method of embodiment 26, further comprising one ormore excipients, carriers, additives, adjuvants or binders in a tabletor capsule.

Embodiment 29. The method of any of embodiments 1 to 28, where thedisorder is selected from the group consisting of Autistic Disorder,Asperger Syndrome, Childhood Disintegrative Disorder and PervasiveDevelopmental Disorder Not Otherwise Specified (PDD-NOS), andPathological Demand Avoidance (PDA).

Embodiment 30. The method of any of embodiments 1 to 28, where thedisorder is selected from the group consisting of Fragile X Syndrome(FXS), Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermidSyndrome, Rett Syndrome, CDKL5 mutations, and X-Linked Infantile SpasmDisorder.

Embodiment 31. The method of any of embodiments 1 to 30, where thecompound is administered either directly or indirectly via thecirculation.

Embodiment 32. The method of any of embodiments 1 to 31, where saidcompound is administered via an oral, intraperitoneal, intravascular,peripheral circulation, subcutaneous, intraorbital, ophthalmic,intraspinal, intracisternal, topical, infusion, implant, aerosol,inhalation, scarification, intraperitoneal, intracapsular,intramuscular, intranasal, buccal, transdermal, pulmonary, rectal, orvaginal route.

Embodiment 33. The method of any of embodiments 1 to 32, where saideffective amount has a lower limit of about 0.001 milligrams perkilogram mass (mg/kg) of the animal and an upper limit of about 100mg/kg.

Embodiment 34. The method of any of embodiments 1 to 33, whereassessment of efficacy is via measurement of phosphorylated ERK (pERK)or phosphorylated Akt (pAkt) in lymphocytes of the animal, wherenormalization of either pERK or pAkt indicates reduction in severity ofsaid disorder.

Embodiment 33. The method of any of embodiments 1 to 33, wherein saidtreatment produces an improvement in a symptom of ASD or NDD as assessedusing one or more clinical tests selected from the group consisting ofThe Rett Syndrome Natural History/Clinical Severity Scale, AberrantBehavior Checklist Community Edition (ABC), Vineland Adaptive BehaviorScales, Clinical Global Impression of Severity (CGI-S), Clinical GlobalImpression Improvement (CGI-I), the Caregiver Strain Questionnaire(CSQ), or one or more physiological tests selected from the groupconsisting of electroencephalogram (EEG) spike frequency, overall powerin frequency bands of an EEG, hemispheric coherence of EEG frequencies,stereotypic hand movement, QTc and heart rate variability (HRV),abnormal cellular expression of Phospho-ERK1/2 and Phospho-Akt, abnormalexpression of growth-associated protein-43 (GAP-43), abnormal expressionof synaptophysin (SYN), respiratory irregularities and coupling ofcardiac and respiratory function compared to control animals notsuffering from said disorder.

Embodiment 36. The method of any of embodiments 1-33, where said symptomof ASD is cognitive impairment or cognitive dysfunction, one or moresigns or symptoms of memory loss, loss of spatial orientation, decreasedability to learn, decreased ability to form short- or long-term memory,decreased episodic memory, decreased ability to consolidate memory,decreased spatial memory, decreased synaptogenesis, decreased synapticstability, deficits in executive function, deficits in cognitive mappingand scene memory, deficits in declarative and relational memory,decreased rapid acquisition of configural or conjunctive associations,decreased context-specific encoding and retrieval of specific events,decreased episodic and/or episodic-like memory, anxiety, abnormal fearconditioning, abnormal social behaviour, repetitive behaviour, abnormalnocturnal behavior, seizure activity, abnormal locomotion, abnormalcellular expression of Phospho-ERK1/2 and Phospho-Akt, and bradycardia.

Embodiment 37. A method for detecting presence of, severity, orevaluation of therapeutic efficacy of any of the preceding embodiments,comprising measuring expression of Phospho-ERK1/2 or Phospho-Akt in aperipheral lymphocyte of a subject with an ASD compared to theexpression of Phospho-ERK1/2 or Phospho-Akt in a peripheral lymphocyteof a group of subjects not having an ASD, or to the expressionPhospho-ERK1/2 or Phospho-Akt in a peripheral lymphocyte of the subjectbefore treatment.

EXAMPLES

The present invention is further illustrated by the following examples.These examples are offered by way of illustration only and are notintended to limit the scope of the invention.

General Methods of Synthesis of Compounds

Flash chromatography was performed using Scharlau 60 (40-60 μm mesh)silica gel. Analytical thin layer chromatography was carried out on 0.20mm pre-coated silica gel plates (ALUGRAM® SIL G/UV₂₅₄) and compoundsvisualized using UV fluorescence, or heating of plates dipped inpotassium permanganate in alkaline solution.

Melting points in degrees Celsius (° C.) were determined on anElectrothermal® melting point apparatus and are uncorrected.

Optical rotations were measured at 20° C. on a Perkin Elmer 341polarimeter using 10 cm path length cells and are given in units of 10⁻¹degcm² g⁻¹. Samples were prepared in the solvent indicated at theconcentration specified (measured in g/100 cm³). IR spectra wererecorded on a Perkin Elmer Spectrum One FT-IR spectrometer. The sampleswere prepared as thin films on sodium chloride discs or as solids inpotassium bromide discs. A broad signal indicated by br. The frequencies(□) as absorption maxima are given in wavenumbers (cm⁻¹).

NMR spectra were recorded on a Broker AVANCE DRX400 (¹H, 400 MHz; ¹³C,100 MHz) or a Broker AVANCE 300 (¹H, 300 MHz; ¹³C, 75 MHz) spectrometerat ambient temperatures. For ¹H NMR data chemical shifts are describedin parts per million downfield from SiMe₄ and are reported consecutivelyas position (5H), relative integral, multiplicity (s=singlet, d=doublet,t=triplet, dd=doublet of doublets, m=multiset, br=broad), couplingconstant (J/Hz) and assignment. For ¹³C NMR data, chemical shifts aredescribed in parts per million relative to CDCl₃ and are reportedconsecutively as position (δ_(C)), degree of hybridization as determinedby DEPT experiments, and assignment. ¹H NMR spectra were referencedinternally using SiMe₄ (δ 0.00) or CDCl₃ (δ 7.26). ¹³C NMR spectra werereferenced internally using CDCl₃ (δ 77.0). When two sets of peaks arisein the NMR spectra due to different conformations around theglycine-proline amide bond, the chemical shift for the minor cisconformer is marked with an asterisk (*).

Accurate mass measurements were recorded on a VG-70SE mass spectrometer.

Hexane and dichloromethane were distilled prior to use. Methanol wasdried using magnesium turnings and iodine, and distilled under nitrogen.Triethylamine was dried over calcium hydride and distilled undernitrogen.

Example 1: Synthesis of(8aS)-Methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclic G-2MeP)

n-BuLi (1.31 M, 4.68 cm³, 6.14 mmol) was added dropwise to a stirredsolution of diisopropylamine (0.86 cm³, 6.14 mmol) in drytetrahydrofuran (10 cm³) at −78° C. under an atmosphere of nitrogen. Thesolution was stirred for 5 min, warmed to 0° C. and stirred for 15 min.The solution was then added dropwise to a solution of oxazolidinone 8(1.00 g, 4.09 mmol) in dry tetrahydrofuran (20 cm³) at −78° C. over 20min (turned to a dark brown colour), stirred for a further 30 min theniodomethane (0.76 cm³, 12.3 mmol) was added dropwise over 5 min. Thesolution was warmed to −50° C. over 2 h. Water (15 cm³) was added andthe solution warmed to room temperature and extracted with chloroform(3×40 cm³). The combined organic extracts were dried (MgSO₄), filteredand evaporated to dryness in vacuo to give a dark brown semi-solid.Purification of the residue by flash column chromatography (15% ethylacetate-hexane) afforded oxazolidinone 9 (0.67 g, 63%) as a pale yellowsolid: mp 55-57° C. (lit., 57-60° C.); (300 MHz, CDCl₃) 1.53 (3H, s,CH₃), 1.72-2.02 (3H, m, Proβ-H and Proγ-H₂), 2.18-2.26 (1H, m, Proβ-H),3.15-3.22 (1H, m. Proδ-H), 3.35-3.44 (1H, m, Proδ-H) and 4.99 (1H, s,NCH).

Methyl L-2-methylprolinate hydrochloride 10

-   -   a) Using acetyl chloride

Oxazolidinone 9 (0.60 g, 2.33 mmol) was dissolved in dry methanol (15cm³) under an atmosphere of nitrogen and acetyl chloride (0.33 cm³, 4.66mmol) was added drop wise to the ice-cooled solution. The solution washeated under reflux for 4.5 h, then the solvent removed under reducedpressure to give a brown oil which was purified by flash columnchromatography (10% CH₃OH—CH₂Cl₂) affording the hydrochloride 10 (0.2 g,48%) as a flaky white solid: mp 107-109° C. (lit., 106-108° C.); δ_(H)(300 MHz, CDCl₃) 1.81 (3H, s, CH₃), 1.93-2.14 (3H, m, Proβ-H_(A)H_(B)and Proγ-H₂), 2.33-2.39 (1H, m, Proβ-H_(A)H_(B)), 3.52-3.56 (2H, in,Proδ-H₂) and 3.82 (3H, s, CO₂CH₃).

b) Using thionyl chloride

An ice-cooled solution of oxazolidinone 9 (53 mg, 0.21 mmol) in drymethanol (1 cm³) was treated dropwise with thionyl chloride (0.045 cm³,0.62 mmol). The solution was heated under reflux for 2.5 h, cooled andthe solvent removed under reduced pressure to yield a brown oil. The oilwas dissolved in toluene (5 cm³), concentrated to dryness to removeresidual thionyl chloride and methanol then purified by flash columnchromatography (10% CH₃OH—CH₂Cl₂) to afford the hydrochloride 10 (16 mg,43%) as a flaky white solid. The ¹H NMR assignments were in agreementwith those reported above.

Methyl-N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 12

Dry triethylamine (0.27 cm³, 1.96 mmol) was added dropwise to a solutionof hydrochloride 10 (0.11 g, 0.61 mmol) and N-benzyloxycarbonyl-glycine11 (98.5%) (0.17 g, 0.79 mmol) in dry dichloromethane (35 cm³) under anatmosphere of nitrogen at room temperature, and the reaction mixturestirred for 10 min. Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl,97%) (0.196 g, 0.77 mmol) was added and the resultant colourlesssolution was stirred for 20.5 h. The solution was washed successivelywith 10% aqueous hydrochloric acid (30 cm³) and saturated aqueous sodiumhydrogen carbonate (30 cm³), dried (MgSO₄), filtered and evaporated todryness in vacuo. Purification of the resultant residue by flash columnchromatography (50-80% ethyl acetate-hexane; gradient elution) yieldeddipeptide 12 (0.18 g, 92%) as a colourless oil. Amide 12 was shown toexist as a 98:2 trans:cis mixture of conformers by ¹³C NMR analysis (theratio was estimated from the relative intensities of the resonances at δ20.8 and 23.5 assigned to the Proγ-C atoms of the minor and majorconformers, respectively): [α]_(D) −33.0 (c 1.0 in MeOH); v_(max)(film)/cm⁻¹ 3406, 2952, 1732, 1651, 1521, 1434, 1373, 1329, 1310, 1284,1257, 1220, 1195, 1172, 1135, 1107, 1082, 1052, 1029, 986, 965, 907,876, 829, 775, 738 and 699; δ_(H) (300 MHz, CDCl₃) 1.49 (3H, s, CH₃),1.77-2.11 (4H, m, Proβ-H₂ and Proγ-H₂), 3.43-3.48 (2H, m, Proδ-H₂), 3.61(3H, s, OCH₃), 3.85-3.89 (2H, in, Glyα-H₂), 5.04 (2H, s, PhCH₂). 5.76(1H, br s, N—H) and 7.21-7.28 (5H, s, ArH); δ_(C) (75 MHz, CDCl₃) 13.8*(CH₃, Proα-CH₃), 21.1 (CH₃, Proα-CH₃), 20.8* (CH₂, Proγ-C), 23.5 (CH₂,Proγ-C), 38.0 (CH₂, Proβ-C), 40.8* (CH₂, Proβ-C), 43.3 (CH₂, Glyα-C),45.5* (CH₂, Glyα-C), 46.6 (CH₂, Proδ-C), 48.7* (CH₂, Proδ-C), 51.9*(CH₃, OCH₃), 52.1 (CH₃, OCH₃), 60.0* (quat., Proα-C), 66.0 (quat.,Proα-C), 66.3 (CH₂, PhCH₂), 68.6* (CH₂, PhCH₃), 127.5 (CH, Ph), 127.6(CH, Ph), 127.9* (CH, Ph), 128.1 (CH, Ph), 128.3* (CH, Ph), 136.2(quat., Ph). 155.9 (quat., NCO₂), 166.0 (quat., Gly-CON), 169.4* (quat.,Gly-CON) and 173.6 (quat., CO₂CH₃); m/z (EI+) 334.1535 (M⁺. C₁₇H₂₂N₂O₅requires 334.1529).

(8aS)-Methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclic G-2MeP)

To a solution of dipeptide 12 (0.167 g, 0.51 mmol) in methanol (8.0 cm³)was added 10% Pd on activated charcoal (8.1 mg, 0.076 mmol) and thevessel flushed with hydrogen gas. The resulting suspension was stirredvigorously under an atmosphere of hydrogen for 15 h. The mixture wasthen filtered through a Celite pad then a short plug of silica gel withmethanol, and the solvent removed under reduced pressure to producecyclic G-2MeP (83 mg, 98%) as a yellow solid: mp 133-135° C.; [α]_(D)−128.1 (c 0.52 in MeOH); δ_(H) (300 MHz, CDCl₃) 1.36 (3H, s, CH₃),1.87-2.01 (3H, m, Proβ-H_(A)H_(B) and Proγ-H₂), 2.07-2.21 (1H, m,Proβ-H_(A)H_(B)), 3.45-3.64 (2H, m, Proδ-H₂), 3.82 (1H, dd, J 17.1 and4.1, CH_(A)H_(B)NH), 3.99 (1H, d, J 17.1, CH_(A)H_(B)NH) and 7.66 (1H,br s, N—H); δ_(C) (75 MHz, CDCl₃) 20.2 (CH₂, Proγ-C), 23.2 (CH₃,Proα-CH₃), 35.0 (CH₂, Proβ-C), 44.7 (CH₂, Proδ-C), 45.9 (CH₂, CH₂NH),63.8 (quat., Proα-C), 163.3 (quat., NCO) and 173.3 (quat., CONH); m/z(EI+) 168.08986 (M⁺. C₈H₁₂N₂O₂ requires 168.08988).

Example 2: Synthesis of(8aS)-Methyl-spiro[cyclohexane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1,4(2H)-dione(Cyclic cyclohexyl-G-2-MeP)

N-benzyloxycarbonyl-1-aminocyclohexane-1-carboxylic acid (14)

To a suspension of 1-aminocyclohexanecarboxylic acid 13 (0.72 g, 5.02mmol) and sodium carbonate (1.6 g, 15.1 mmol) were dissolved inwater-dioxane (21 cm³, 3:1) was added benzyl chloroformate (0.79 cm³,5.52 mmol) was added drop wise and the solution was stirred at roomtemperature for 19.5 h. The aqueous layer was washed with diethyl ether(60 cm³), acidified with 2 M HCl and extracted with ethyl acetate (2×60cm³). The organic layers were combined, dried (MgSO₄), filtered andevaporated under reduced pressure to produce a colourless oil, whichsolidified on standing to crude carbamate 14 (1.23 g, 88%) as a whitesolid: mp 152-154° C. (lit., 148-150° C.); 4 (400 MHz, CDCl₃) 1.27-1.56(3H, m, 3× cyclohexyl-H), 1.59-1.73 (3H, m, 3× cyclohexyl-H), 1.85-1.91(2H, m, 2× cyclopentyl-H), 2.05-2.09 (2H, m, 2× cyclopentyl-H), 5.02(1H, br s, N—H), 5.12 (2H, s, OCH₂Ph) and 7.27-7.36 (5H, s, Ph); 4(100MHz, CDCl₃) 21.1 (CH₂, 2× cyclohexyl-C), 25.1 (CH₂, 2× cyclohexyl-C),32.3 (CH₂, cyclohexyl-C), 59.0 (quat., 1-C), 67.1 (CH₂, OCH₂Ph), 128.1(CH, Ph), 128.2 (CH, Ph), 128.5 (CH, Ph), 136.1 (quat., Ph), 155.7(quat., NCO₂) and 178.7 (quat., CO₂H).

Methyl-N-benzyloxycarbonyl-cyclohexyl-glycyl-L-2-methylprolinate (15)

Dry triethylamine (0.21 cm³, 1.5 mmol) was added dropwise to a solutionof hydrochloride 10 (84.0 mg, 0.47 mmol), carboxylic acid 14 (0.17 g,0.61 mmol) and 1-hydroxy-7-azabenzotriazole (16 mg, 0.12 mmol) in dry1,2-dichloroethane (26 cm³) under an atmosphere of nitrogen at roomtemperature, and the reaction mixture stirred for 10 min.2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (0.13 g, 0.47mmol) was added and the resultant solution heated under reflux for 21 h,then washed successively with 10% aqueous hydrochloric acid (30 cm³) andsaturated aqueous sodium hydrogen carbonate (30 cm³), dried (MgSO₄),filtered and evaporated to dryness in vacuo. Purification of theresultant residue by flash column chromatography (40-50% ethylacetate-hexane; gradient elution) yielded amide 15 (16 mg, 9%) as awhite solid. Amide 15 was shown to exist as a 11:1 trans:cis mixture ofconformers by ¹³C NMR analysis (the ratio was estimated from therelative intensities of the resonances at δ 41.3 and 48.2 assigned tothe Proδ-C atoms of the minor and major conformers, respectively): mp219-222° C.; [α]_(D) −44.9 (c 1.31 in CH₂Cl₂); v_(max) (film)/cm⁻¹ 3239,2927, 1736, 1707, 1617, 1530, 1450, 1403, 1371, 1281, 1241, 1208, 1194,1165, 1150, 1132, 1089, 1071, 1028, 984, 912, 796, 749, 739 and 699;δ_(H) (400 MHz, CDCl₃) 1.24-2.10 (17H, m, Proα-CH₃, Proβ-H₂, Proγ-H₂ and5× cyclohexyl-H₂), 3.25-3.48 (1H, br m, Proδ-H_(A)H_(B)), 3.61-3.87 (4H,br m, OCH₃ and Proδ-H_(A)/H_(B)). 4.92-5.19 (3H, m, N—H and OCH₂Ph) and7.35-7.37 (5H, s, Ph); δ_(C) (100 MHz, CDCl₃) 21.26 (CH₂, cyclohexyl-C),21.33 (CH₂, cyclohexyl-C). 21.7 (CH₃, Proα-CH₃), 24.8 (CH₂,cyclohexyl-C), 25.0 (CH₂, Proγ-O, 29.4* (CH₂, cyclohexyl-C), 29.7* (CH₂,cyclohexyl-C), 31.1 (CH₂, cyclohexyl-C), 31.6 (CH₂, cyclohexyl-C), 31.9*(CH₂, cyclohexyl-C), 32.2* (CH₂, cyclohexyl-C), 32.8* (CH₂,cyclohexyl-C), 37.3 (CH₂, Proβ-C), 41.4* (CH₂, Proδ-C), 48.2 (CH₂,Proδ-C), 52.1 (CH₃, OCH₃), 59.1 (quat., Glyα-C), 66.7 (CH₂, OCH₂Ph),67.3* (CH₂, OCH₂Ph), 67.4 (quat., Proα-C), 128.0* (CH, Ph), 128.1* (CH,Ph). 128.3 (CH, Ph), 128.5 (CH, Ph), 128.7 (CH, Ph), 136.6 (quat., Ph),153.7 (quat., NCO₂), 171.0 (quat., Gly-CO) and 174.8 (quat., CO₂CH₃);m/z (EI+) 402.2151 (M*. C₂₂H₃₀N₂O₅ requires 402.2155).

(8aS)-Methyl-spiro[cyclohexane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1,4(2-dione(Cyclic cyclohexyl-G-2MeP)

To a solution of amide 15 (40 mg, 0.01 mmol) in methanol (3.3 cm³) wasadded 10% Pd on activated charcoal (1.6 mg, 0.015 mmol) and the vesselflushed with hydrogen gas. The resulting suspension was stirredvigorously under an atmosphere of hydrogen for 61.5 h, then filteredthrough a Celite™ pad with methanol (15 cm³). The filtrate wasconcentrated to dryness under reduced pressure to produce a yellowsemi-solid which was purified by reverse-phase C18 flash columnchromatography (0-10% CH₃CN/H₂O; gradient elution) to produce cycliccyclohexyl-G-2MeP (19 mg, 81%) as a white solid: mp 174-177° C.; [α]_(D)−63.8 (c 1.13 in CH₂Cl₂); v_(max) (film)/cm⁻¹ 3215, 2925, 2854, 1667,1646, 1463, 1427, 1276, 1232, 1171, 1085, 1014, 900, 868, 818, 783, 726and 715; δ_(H) (400 MHz, CDCl₃) 1.31-1.89 (12H, m, 9× cyclohexyl-H and8a-CH₃), 1.94-2.15 (4H, m, 7-H₂ and 8-H₂), 2.26 (1H, td, J 13.7 and 4.5,1× cyclohexyl-H), 3.44-3.51 (1H, m, 6-H_(A)H_(B)), 3.79-3.86 (1H, m,6-H_(A)H_(B)) and 6.40 (1H, hr s, N—H); δ_(C) (100 MHz, CDCl₃) 19.5(CH₂, 7-C), 20.6 (CH₂, cyclohexyl-C), 20.8 (CH₂, cyclohexyl-C), 24.5(CH₂, cyclohexyl-C), 25.0 (CH₃, 8a-CH₃), 33.7 (CH₂, cyclohexyl-C), 36.3(CH₂, 8-C), 36.5 (CH₂, cyclohexyl-C), 44.7 (CH₂, 6-C), 59.5 (quat.,8a-C), 64.0 (quat., 3-C), 168.1 (quat., 4-C) and 171.6 (quat., 1-C); m/z(EI+) 236.15246 (M⁺. C₁₃H₂₀N₂O₂ requires 236.15248).

Example 3: Synthesis of(8aS)-Allyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (CyclicG-2-AllylP)

(2R,5S)-4-Allyl-2-trichloromethyl-1-aza-3-oxabicyclo[3.3.0]octan-4-one17

n-BuLi (1.31 M, 9.93 cm³, 13.0 mmol) was added dropwise to a stirredsolution of diisopropylamine (1.82 cm³, 13.0 mmol) in drytetrahydrofuran (20 cm³) at −78° C. under an atmosphere of nitrogen. Thesolution was stirred for 5 min, warmed to 0° C., stirred for 15 min thenadded dropwise to a solution of pro-oxazolidinone 16 (2.12 g, 8.68 mmol)in dry tetrahydrofuran (40 cm³) at −78° C. over 20 min and the reactionmixture was stirred for a further 30 min then allyl bromide (2.25 cm³,26.0 mmol) was added dropwise over 5 min. The solution was warmed slowlyto −30° C. over 4 h, quenched with H₂O (30 cm³) and the mixture warmedto room temperature and extracted with chloroform (3×80 cm³). Thecombined organic extracts were dried (MgSO₄), filtered and evaporated todryness in vacuo to produce a dark brown semi-solid which was purifiedby flash column chromatography (10-20% ethyl acetate-hexane; gradientelution) to produce oxazolidinone 17 (1.48 g, 60%) as an orange oilwhich solidified at 0° C., for which the nmr data were in agreement withthat reported in the literature: δ_(H) (400 MHz, CDCl₃) 1.58-1.92 (2H,m, Proγ-H₂), 1.96-2.14 (2H, m, Proβ-H₂), 2.50-2.63 (2H, m, Proδ-H₂),3.12-3.23 (2H, m, CH₂—CH═CH₂), 4.97 (1H, s, NCH), 5.13-5.18 (2H, m,CH═CH₂) and 5.82-5.92 (1H, m, CH═CH₂); δ_(C) (100 MHz, CDCl₃) 25.1 (CH₂,Proγ-C), 35.1 (CH₂, Proβ-C), 41.5 (CH₂, Proδ-C), 58.3 (CH₂, CH₂CH═CH₂),71.2 (quat., Proα-C), 100.4 (quat., CCl₃), 102.3 (CH, NCH), 119.8 (CH₂.CH₂CH═CH₂), 131.9 (CH, CH₂CH═CH₂) and 176.1 (quat., C═O); m/z (CI+)284.0009 [(M+H)⁺. C₁₀H₁₃ ³⁵Cl₃NO₂ requires 284.0012], 285.9980 [(M+H)⁺.C₁₀H₁₃ ³⁵Cl₂ ³⁷ClNO₂ requires 285.9982], 287.9951 [(M+H)⁺. C₁₀H₁₃³⁵Cl³⁷NO₂ requires 287.9953] and 289.9932 [(M+H)⁺. C₁₀H₁₃ ³⁷Cl₃NO₂requires 289.9923].

Methyl L-2-allylprolinate hydrochloride 18

An ice-cooled solution of oxazolidinone 17 (0.64 g, 2.24 mmol) in drymethanol (15 cm³) was treated drop wise with a solution of acetylchloride (0.36 cm³, 5.0 mmol) in methanol (5 cm³). The solution washeated under reflux for 24 h, then cooled and tire solvent removed underreduced pressure. The resultant brown oil was dissolved in toluene (40cm³) and concentrated to dryness to remove residual thionyl chloride andmethanol, then purified by flash column chromatography (5-10%CH₃OH—CH₂Cl₂; gradient elution) to afford hydrochloride 18 (0.29 g, 63%)as a green solid for which the NMR data were in agreement with thatreported in the literature: δ_(H) (300 MHz, CDCl₃) 1.72-2.25 (3H, m,Proβ-H_(A)H_(B) and Proγ-H₂), 2.32-2.52 (1H, m, Proβ-H_(A)H_(B)),2.72-3.10 (2H, m, Proδ-H₂), 3.31-3.78 (2H, m, CH₂CH═CH₂), 3.84 (3H, s,CO₂CH₃). 5.20-5.33 (2H, m, CH═CH₂), 5.75-5.98 (1H, m, CH═CH₂) and 8.06(1H, br s, N—H); m/z (CI+) 170.1183 [(M+H)⁴. C₉H₁₆NO₂ requires170.1181].

Methyl-N-tert-butyloxycarbonyl-glycyl-L-2-allylprolinate 20

Dry triethylamine (0.28 cm³, 2.02 mmol) was added dropwise to a solutionof hydrochloride 18 (0.13 g, 0.63 mmol) andN-tert-butyloxycarbonyl-glycine 19 (0.14 g, 0.82 mmol) in drydichloromethane (35 cm³) under an atmosphere of nitrogen at roomtemperature, and the reaction mixture was stirred for 10 min.Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) (0.20 g, 0.80mmol) was added and the solution stirred for 19.5 h, then washedsuccessively with 10% aqueous hydrochloric acid (33 cm³) and saturatedaqueous sodium hydrogen carbonate (33 cm³), dried (MgSO₄), filtered andevaporated to dryness in vacuo. Purification of the resultant residue byflash column chromatography (40% ethyl acetate-hexane) yielded dipeptide20 (0.09 g, 45%) as a light yellow oil: [α]_(D) +33.8 (c 0.83 inCH₂Cl₂); v_(max) (film/cm⁻¹ 3419, 3075, 2977, 2930, 2874, 1739, 1715,1656, 1499, 1434, 1392, 1366, 1332, 1268, 1248, 1212, 1168, 1122, 1051,1026, 1003, 943, 919, 867, 830, 779, 739, 699 and 679; δ_(H) (300 MHz,CDCl₃) 1.42 [9H, s, C(CH₃)₃], 1.93-2.08 (4H, m, Proβ-H₂ and Proγ-H₂),2.59-2.67 (1H, m, CH_(A)H_(B)CH═CH₂), 3.09-3.16 (1H, m,CH_(A)H_(B)CH═CH₂), 3.35-3.44 (1H, m, Proδ-H_(A)H_(B)), 3.56-3.62 (1H,m, Proδ-H_(A)H_(B)), 3.70 (3H, s, OCH₃), 3.89 (2H, d, J 4.2, Glyα-H₂),5.06-5.11 (2H, m, CH═CH₂), 5.42 (1H, br s, Gly-NH) and 5.58-5.72 (1H, m,CH═CH₂); δ_(C) (75 MHz, CDCl₃) 23.7 (CH₂, Proγ-C), 28.3 [CH₂, C(CH₃)₃],35.0 (CH₂, Proβ-C), 37.6 (CH₂, CH₂CH═CH₂), 43.3 (CH₂, Glyα-C), 47.5(CH₂, Proδ-C), 52.5 (CH₃, OCH₃), 68.8 (quat, Proα-C), 79.5 [quat.,C(CH₃)₃], 119.4 (CH₂, CH═CH₂), 132.9 (CH, CH═CH₂), 155.7 (quat., NCO₂),166.9 (quat., Gly-CON) and 173.8 (quat., CO₂CH₃); m/z (EI+) 326.1845(M⁺. C₁₆H₂₆N₂O₅ requires 326.1842).

(8aS)-Allyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclic G-2AllylP)

To a solution of dipeptide 20 (0.09 g, 0.28 mmol) in dichloromethane (9cm³) at room temperature was added trifluoroacetic acid (1 cm³, 0.013mmol) dropwise and the reaction mixture was stirred for 1 h under anatmosphere of nitrogen. The solution was evaporated under reducedpressure to give a colorless oil which was dissolved in dichloromethane(10 cm³), dry triethylamine (0.096 cm³, 0.69 mmol) was added and thereaction mixture stirred for 4.5 h, after which further triethylamine(0.0% cm³, 0.69 mmol) was added. The reaction mixture was stirredovernight, concentrated to dryness to give a green oil which waspurified by flash column chromatography (10% CH₃OH—CH₂Cl₂) to producecyclic G-2AllylP (20 mg, 37%) as an off-white solid: mp 106-109° C.;[α]_(D) −102.7 (c 0.95 in CH₂Cl₂); v_(max) (CH₂Cl₂)/cm⁻¹ 3456, 3226,2920, 1666, 1454, 1325, 1306, 1299, 1210, 1133, 1109, 1028, 1010, 949,928, 882, 793, 761 and 733; δ_(H)(400 MHz, CDCl₃) 1.92-2.01 (2H, m,Proγ-H₂), 2.09-2.16 (2H, m, Proβ-H₂). 2.39-2.56 (2H, m, CH₂CH₂═CH₂),3.46-3.53 (1H, m, Proδ-H_(A)H_(B)). 3.78-3.87 (2H, m, Proδ-H_(A)H_(B)and Glyα-H_(A)H_(B)). 4.09 (1H, d, J 17.2, Glyα-H_(A)H_(B)), 5.16-5.20(2H, m, CH═CH₂), 5.73-5.84 (1H, m, CH═CH₂) and 7.17 (1H, br s, N—H);δ_(C) (100 MHz, CDCl₃) 20.1 (CH₂, Proγ-C), 34.1 (CH₂, Proβ-C), 41.7(CH₂, CH₂CH₂═CH₂), 44.9 (CH₂, Proδ-C), 46.4 (CH₂, Glyα-C), 67.2 (quat.,Proα-C), 120.9 (CH₂, CH═CH₂), 131.0 (CH, CH═CH₂), 163.4 (quat., NCO) and171.7 (quat., CONH); m/z (EI+) 195.1132 (M⁺. C₁₀H₁₅N₂O₂ requires195.1134).

Example 4: Synthesis of(8aS)-Methyl-spiro[cyclopentane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1,4(2H)-dione(Cyclic Cyclopentyl-G-2-MeP)

N-Benzyloxycarbonyl-1-aminocyclopentane-1-carboxylic acid 21

A solution of benzyl chloroformate (0.290 g, 1.1 mmol) in dioxane (2.5cm³) was added dropwise to a solution of 1-aminocyclopentanecarboxylicacid (Fluka) (0.2 g, 1.54 mmol) and sodium carbonate (0.490 g, 4.64mmol) in water (5 cm³) at 0° C. Stirring was continued at roomtemperature overnight and the reaction mixture washed with ether. Theaqueous layer was acidified with 2M hydrochloric acid, extracted withethyl acetate, dried (Na₂SO₄), filtered and the solvent removed toafford carbamate 21 (0.253 g, 62%) as an oil which solidified onstanding. Carbamate 21 was shown to be a 70:30 mixture of conformers by¹H NMR analysis (the ratio was estimated from the integration of theresonances at δ 5.31 and 7.29-7.40, assigned to the N—H protons of themajor and minor conformers, respectively): mp 70-80° C. (lit.¹ 82-86°C., ethyl acetate, petroleum ether); δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.83(4H, br s, 2× cyclopentyl-H₂), 2.04 (2H, br s, cyclopentyl-H₂),2.20-2.40 (2H, m, cyclopentyl-H₂), 5.13 (2H, br s, OCH₂Ph), 5.31 (0.7H,br s, N—H) and 7.29-7.40 (5.3H, m, Ph and N—H*); δ_(C) (100 MHz; CDCl₃)24.6 (CH₂, cyclopentyl-C), 37.5 (CH₂, cyclopentyl-C), 66.0 (quat.,cyclopentyl-C), 66.8 (CH₂, OCH₂Ph), 128.0 (CH, Ph), 128.1 (CH, Ph),128.4 (CH, Ph), 136.1 (quat, Ph), 155.8 (quat., NCO₂) and 179.5 (quat.,CO₂H). * denotes resonance assigned to minor conformer.

Methyl N-benzyloxycarbonyl cyclopentyl-glycyl-L-2-methylprolinate 22

Dry triethylamine (0.19 cm³, 1.4 mmol) was added dropwise to a solutionof hydrochloride 10 (78 mg, 0.43 mmol), carboxylic acid 21 (0.15 g, 0.56mmol) and 1-hydroxy-7-azabenzotriazole (Acros) (15 mg, 0.11 mmol) in dry1,2-dichloroethane (24 cm³) under an atmosphere of nitrogen at roomtemperature, and the reaction mixture stirred for 10 min.2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) (Aldrich)(0.12 g, 0.43 mmol) was added and the resultant solution heated underreflux for 19 h, then washed successively with 10% aqueous hydrochloricacid (30 cm³) and saturated aqueous sodium hydrogen carbonate (30 cm³),dried (MgSO₄), filtered and evaporated to dryness in vacuo. Purificationof the resultant residue by flash column chromatography (60% ethylacetate-hexane) yielded amide 22 (39 mg, 23%) as a white solid. Amide 22was shown to exist as a 3:1 trans:cis mixture of carbamate conformers by¹³C NMR analysis (the ratio was estimated from the relative intensitiesof the resonances at δ 154.1 and 155.7 assigned to the carbamatecarbonyl-C atoms of the major and minor conformers, respectively): mp200-203° C.; [α]_(D) −54.5 (c 1.52 in CH₂Cl₂); v_(max) (film)/cm⁻¹ 3432,3239, 3042, 2953, 1736, 1712, 1627, 1540, 1455, 1417, 1439, 1374, 1282,1256, 1216, 1194, 1171, 1156, 1136, 1100, 1081, 1042, 1020, 107, 953,917, 876, 756 and 701; δ_(H) (400 MHz, CDCl₃) 1.33-1.53 (3H, br m,Proα-CH₃), 1.62-2.20 (11H, m, Proβ-H₂, Proγ-H₂ and 7× cyclopentyl-H),2.59-2.71 (1H, br m, 1× cyclopentyl-H), 3.31-3.42 (1H, br m,Proδ-H₂H_(B)), 3.58-3.79 (4H, br m, OCH₃ and Proδ-H_(A)H_(B)), 4.92-5.17(3H, m, N—H and OCH₂Ph) and 7.27-7.42 (5H, s, Ph); δ_(C) (100 MHz,CDCl₃) 21.7 (CH₂, Proα-CH₃), 24.1* (CH₂, cyclopentyl-C), 24.2 (CH₂,cyclopentyl-C), 24.4 (CH₂, Proγ-C), 24.5 (CH₂, cyclopentyl-C), 36.4(CH₂, cyclopentyl-C), 37.1 (CH₂, cyclopentyl-C), 37.2* (CH₂,cyclopentyl-C), 37.7 (CH₂, Proβ-C), 38.2* (CH₂, cyclopentyl-C), 48.5(CH₂, Proδ-C), 52.1 (CH₂, OCH₃), 66.6 (CH₂, OCH₂Ph), 66.9 (quat.,Proα-C), 67.2 (quat., Glyα-C), 127.8 (CH, Ph), 128.2 (CH, Ph), 128.4(CH, Ph), 136.6 (quat., Ph), 154.1 (quat., NCO₂), 155.7* (quat., NCO₂),170.5 (quat., Gly-CO) and 174.7 (quat., CO₂CH₃); m/z (EI+) 388.1991 (M⁺.C₂₁H₂₈N₂O₅ requires 388.1998).

(8aS)-Methyl-spiro[cyclopentane-1,3(4H)-tetrahydropyrrolo[1,2-a]pyrazine]-1,4(2H)-dione

(Cyclic cyclopentyl-G-2MeP)

To a solution of amide 22 (54 mg, 0.14 mmol) in methanol (4.6 cm³) wasadded 10% Pd on activated charcoal (2.2 mg, 0.021 mmol) and the vesselflushed with hydrogen gas. The resulting suspension was stirredvigorously under an atmosphere of hydrogen for 17 h, then filteredthrough a Celite™ pad with methanol (15 cm³). The filtrate wasconcentrated to dryness under reduced pressure to give a yellowsemi-solid which was purified by reverse-phase C18 flash columnchromatography (0-10% CH₃CN/H₂O; gradient elution) to afford cycliccyclopentyl-G-2MeP (20 mg, 65%) as a yellow solid: mp 160-163° C.;[α]_(D) −97.9 (c 1.61 in CH₂Cl₂); v_(max) (film)/cm⁻¹ 3429, 2956, 2928,2856, 1667, 1643, 1463, 1432, 1373, 1339, 1254, 1224, 1175, 1086, 1048,976, 835, 774 and 730; δ_(H) (300 MHz, CDCl₃) 1.47 (3H, hr s, 8a-CH₃),1.56-2.19 (11H, m, 8-H₂, 7-H₂ and 7× cyclopentyl), 2.58-2.67 (1H, hr m,1× cyclopentyl), 3.48-3.56 (1H, m, 6-H_(A)H_(B)), 3.72-3.82 (1H, m,6-H_(A)H_(B)) and 6.56 (1H, br s, N—H); 4: (75 MHz, CDCl₃) 19.9 (CH₂,7-C), 24.6 (CH₂, cyclopentyl), 24.92 (CH₃, 8a-CH₃), 24.93 (CH₂,cyclopentyl), 36.0 (CH₂, 8-C), 38.7 (CH₂, cyclopentyl), 41.9 (CH₂,cyclopentyl), 44.8 (CH₂, 6-C), 64.3 (quat., 8a-C), 66.8 (quat., 3-C),168.3 (quat., 4-C) and 172.2 (quat, 1-C); m/z (EI+) 222.1369 (M⁺.C₁₂H₁₈N₂O₂ requires 222.1368).

In Vitro and In Vivo Testing

The following pharmacological studies demonstrate efficacy of cyclicG-2-AllylP in attenuation of cognitive impairment. They are not intendedto be limiting, and other compositions and methods of this invention canbe developed without undue experimentation. All of those compositionsand methods are considered to be part of this invention. All thefollowing experiments were carried out using protocols developed underguidelines approved by the University of Auckland Animal EthicsCommittee or comparable regulatory bodies.

Efficacy of nootropic drugs can be conveniently tested using models ofcholinergic hypofunction. Cholinergic hypofunction has been shown tocontribute to dementia-related cognitive decline and remains a target oftherapeutic intervention for Alzheimer's disease (Hunter 2004). Thecholinergic hypofunction model is also applicable to other conditions.For example, it has been shown that scopolamine-induced cholinergichypofunction can selectively impair the recognition accuracy of disgustand anger facial expressions rendering the effect of scopolamine onemotion-recognition similar to those found in Huntington's diseasepatients (Kamboy 2006). Scopolamine has been commonly used to inducecholinergic hypofunction, and is a well-known model for humanAlzheimer's disease, aging and other disorders of cognitive function(Liskowsky et at, Int. J. Dev. Neurosci, 24(2-3): 149-156 (2006),Lindner et al., Psychopharmacology (Berl.) September 27 (2006), Bougeret al., Eur. Neuropsychopharmacol 15(3):331-346 (2005), Ebert et al,Eur. J. Clin. Invest., 28(11):944-949 (1998), Barker et al, Int. J.Geriatr. Psychiatry, 13(4):244-247 (1998), G. Smith, Brain Res.471(2):103-118 (1998), Flood et al, Behav. Neural. Biol.45(2):169-184(1986)).

Example 5: Morris Water Maze (MWM) Model of Learning and Memory Used toAssess Effects of Cyclic G-2-AllylP on Cognitive Function

The purpose of the study was to investigate cyclic G-2AllylP in modes ofcognitive deficit and affective state (anxiety).

Methods

The first part of the study involved acute testing of cG-2-AllylP in theMorris Water Maze Memory (MWM) model. The MWM test is one of the mostfrequently used tests for assessing spatial memory in rats and is wellrecognized to accurately predict effects of disease and treatment onspatial memory generally. Therefore, the MWM test reflects effects ofdisease and treatment in human subjects.

The standard procedure for MWM was followed. We used a circular swimmingpool (80 cm depth×150 cm diameter) filled with opaque water, with thetemperature maintained at 20° C. A platform was hidden 1 cm below thewater surface, with a white flag (10 cm×10 cm) located either 20 cmabove the platform for the visual cue and at 3 o'clock position inrelation to the starting location for a spatial cue. On days 1-4 of theexperiment rats underwent memory acquisition trials with 6 trials (60seconds each) in each day of testing (habituation phase). Latency toreach the platform was recorded and the daily reduction of averagelatency was used to measure the capability to learn where the hiddenplatform was.

On day 3 of the experiment normal, non-aged Wistar rats were split intogroups to receive either saline (n=28) or scopolamine (0.3 mg/kg, i.p.,n=27) to induce memory deficit. Scopolamine 3 was administered half anhour before the probe test commenced.

10 min following the scopolamine treatment, the cyclic G-2AllylP wasadministered orally at 30 mg/kg (n=31) with vehicle-treated animalsadministered the diluent by oral gavage using an identical treatmentprotocol (n=24).

Acute effects of cG-2-AllylP were then tested in animals withscopolamine-induced memory impairment and in age-matched control animalswith no memory impairment to determine any direct pharmacological effecton memory processing. Experimental groups are detailed in the Table 4below.

TABLE 4 Animals Used to Test Effects of cG-2-AllylP on MemoryScopolamine Vehicle Vehicle N = 12 N = 12 cG-2-AllylP N = 15 N = 16

On day 3, the probe MWM test was performed with the platform removed.There were 6 trials, each of maximum duration of 60s, at least 3 minrest between trials). The amount of tune the rats spend swimming nearthe platform provided a measure of how much they relied on visual andspatial cue to locate the platform, as opposed to using a non-spatialstrategy. Data was collected and analysed using Any-maze (v4.2)software.

The data generated from behavioural tests was analysed using one-wayANOVA for determining the difference between the aged-groups. Two-wayANOVA was used for examining the progress of behavioral results with thetime points treated as dependent factors. GraphPad Prism version 3.02was used for data analysis.

Results

Treatment with scopolamine significantly impaired acquisition of spatialmemory in treated animals (time to platform approximately 208% ofcontrol on day 4). Cyclic G-2AllylP (30 mg/kg; daily) significantlyreversed the cognitive impairment induced by scopolamine (FIGS. 1A, 1B,1C).

Example 6: cG-2-AllylP Improves Synaptic Plasticity and Aging-RelatedMemory Loss

Methods

Aged rats (male Wistar rats, 18-20 months old) were divided into fourgroups: two vehicle-treated (groups 1 and 3) and two G-2-AllylP treated(groups 2 and 4) (all groups n=6-8). Cyclic G-2-AllylP was synthesisedby the Department of Medicinal Chemistry and dissolved in normal salinebefore the treatment. On day 1 a single dose of cyclic G-2-AllylP wasgiven centrally (20 ng/animal, i.c.v.) to the animals in groups 2 and 4;saline was administered to groups 1 and 3. The memory tests using NovelObject Recognition Test started either on day 3 (groups 1 and 2) or 24(groups 3 and 4) after the treatment. On the completion of the NORT, therats were killed with an overdose of sodium pentobarbital and wereperfused transcardially with normal saline followed by 10% formalin.Tissues collected at day 7 in groups 1 and 2, and at day 28 from groups3 and 4. The brains were kept in the same fixative for a minimum of 2days before being processed using a standard paraffin embeddingprocedure. Briefly, small blocks (10×10×3 mm) of tissue were fixed forup to 24 hrs. The blocks were then infiltrated and embedded withparaffin and cut in ribbons and mounted on slides. Slides were thenstored until immunostaining was commenced. Synaptogenesis in braintissue was examined using immunohistochemical staining.

Novel Object Recognition Test (NORT)

Exploratory activity is a typical learning behaviour displayed byanimals including humans and rats in novel environments. Exploratoryactivity decreases over time when the novel becomes familiar and thehabituation occurs. In familiar environments, exploratory activity canbe reactivated by introducing a novel object. The increase in exploringbehaviour once the environment is altered following a habituationprovides a measure of the memory for the familiarity and the recognitionof the novelty.

In this Example, we carried out two NORTs, one at days 3-6 and the otherat 24-27 days. The rats were allowed to familiarise themselves with thetesting arena (90×60×40 cm) in the first day of NORT. In the followingtwo days of each test, four novel objects were placed into the testingarena and the rats had 2 trials each day (each of 15 min duration and 2hours apart). The time spent on exploring the objects was reduced oncethe animal tested learned about the objects (training phase). In thelast day (day 4 of each test), one familiar object was replaced by anovel object before the second trial (test 6, testing phase). Theaverage time spent on exploring the 3 familiar objects and the timespent on exploration of the novel object was used as a measure for thememory of familiarity and the novelty recognition.

Effects of cG-2-AllylP on Expression of NMDA Receptors, AMPA Receptors,rKrox-24 and Synaptophysin mRNA in the Hippocampus

It is accepted that the hippocampal formations in humans and animalsplay a crucial role in a number of memory types (Morris et al. 2006Europ. J. Neurosci. 23, 2829). The specific functionality remains underdispute, but there is an understanding that hippocampus plays a key rolein the automatic encoding and initial storage of attended experiences(episodic memory formation), memory consolidation and novelty detection.The first aspect, encoding and short-term storage of memories, isdependent on the synaptic plasticity and synaptic transmission, both ofwhich are linked to glutamatergic neurotransmission.

Glutamatergic transmission is facilitated by two types of glutamatereceptors: N-methyl-D-aspartate receptors (NMDAR) anda-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid receptors AMPA ornon-NMDA receptors.

APMA receptor subunit GluR1 is a post-synaptic receptor and has beencommonly used for memory measurement. GluR1 is believed to mediatecalcium influx, and has a vital function in synaptic plasticity relatedto learning. It has been previously suggested (Hayashi et al., 2000)that incorporation of GluR1 into synapses might be important forlong-term potentiation (LTP), which is essential for learning andmemory.

It had been demonstrated that NMDA receptor subunit NR1 is crucial forformation of spatial memory. In knock-out models where the R1 subunit ofthe NMDA receptor in the pyramidal cells of the CA1 region wasselectively knocked-out, the long-term potentiation was shown to beabolished (Tsien 1996).

Synaptophysin is a presynaptic vesicle protein. Its quantitativedetection is established as a molecular marker of synaptic density.

The neuronal transcript factor Krox24 staining is used as a marker forneuronal plasticity. The protein products of the Krox24 family (as wellas by brain-derived neurotrophic factor, BDNF) have recently linked withstabilizing synaptic modifications occurring duringNMDA-receptor-mediated hippocampal LTP and LTD. (Dragunow. 2006.Behaviour genetics. 23; 293).

Immunohistochemical Staining

Conventional deparaffinisation and rehydration techniques were used toallow the water-based buffers and antibodies to penetrate the tissueslices. Antigen retrieval was used only prior to AMPA receptor (GluR1)staining, i.e. the slides were placed in boiling citrate buffer andallowed to cool.

The following antibodies were used:

-   -   i) primary rabbit antibody to NMDA NR1 subunit, at 1:200        concentration in buffer, incubated for 48 hrs (Chemicon—AB1516),        followed by Sigma fluorescent secondary antibody (alexaFluor        394), at 1:200 dilution, incubated for 24 hours at 40C.    -   ii) primary antibody to AMPA GluR1 subunit, at 1:30        concentration in buffer, incubated for 48 hrs (Chemicon—AB1504)        followed by 3,3′-diaminobenzidine (DAB) at 1:200 dilution,        incubated for 24 hours at 40C.    -   iii) Primary antibody to mSynaptophysin (Sigma—S5768), at 1:200        concentration in buffer, followed by DAB at 1:200 dilution,        incubated for 24 hours at 40C.    -   iv) Primary antibody to rKrox-24 (Santa Cruz—catalogue number        SC-189) at 1:200 concentration in the buffer, followed by        anti-rabbit secondary antibody at concentration of 1:200        dilution, incubated for 24 hours at 40C.    -   y) Antibodies were detected using light microscopy.

Results

NORT

A trend to improve the novelty recognition in the groups treated withcG-2-AllylP was observed after 27 days (FIG. 2), but not 6 days afterthe treatment (no figure). We conclude that the cG-2AllylP treatmentimproved novelty recognition in the drug-treated animals at 27 days.

AMPA Glutamate Receptor-1 Staining

Hippocampal slices from regions CA1 (granular cell layer, strata oriensand radiatum) and CA3 (pyramidal cell layer) were stained for AMPAreceptors GluR1.

In CA3 there was no change in the number of receptors in each region oneither day 7 or 28. There was however a significant increase in thenumber of AMPA receptors in CA1 (granular cell layer) (FIG. 4) and CA1stratum oriens (FIG. 5) and on day 28.

That histological change was correlated with the improved performance inthe novel object recognition test. The improved memory (FIG. 2) wascorrelated to the elevated AMPA glutamate receptor-1 (FIG. 3). Weconcluded that cG-2-AllylP improved glutamatergic neurotransmission(GluR1) at post-synaptic level.

We observed that cG-2-AllylP treatment resulted in a long term increasein GluR1 staining on the post-synapses and increased the density ofpre-synaptic vesicles. As the majority of vesicles in the hippocampusare glutamic vesicles, we concluded that the long term memoryimprovement was associated with increased glutamic neurotransmission.

Synaptophysin Staining

We subsequently analysed effect of cG-2AllylP on the levels ofsynaptophysin staining in CA3 and CA1 regions of the hippocampus.

In all tested areas there was either a significant increase (CA3) or aclear trend towards (CA1—strata oriens and radiatum) the increase in thedensity of synaptophysin staining at 28 days post-treatment. Thatincrease is a marker of increased synaptic plasticity and a clearindication of synaptogenesis which is a most likely cause of theimprovement in the performance of the treated groups in applied memorytests.

NMDA Receptor-1 Staining

While there is a significant improvement in AMPA receptorspost-treatment, the changes in the NMDA receptors are not so pronounced(FIGS. 9A, 9B and 9C).

Krox24 Staining

We analysed the density of the Krox24 staining in the CA1-2 regions ofthe hippocampus. We observed a trend towards the increased density intreatment group in comparison to the vehicle treated group. We concludethat the Krox24 staining results positively correlate with improvedmemory function (FIG. 10).

Example 7: cG-2-AllylP Increases the Number in Pre-Synaptic Vesicles inthe Hippocampus of Middle Aged Rats

Methods

Four middle aged Wistar male rats (12 months) were divided into twogroups: one vehicle-treated (n=2) and one cG-2-AllylP-treated (n=2). Therats were treated subcutaneously with 3 mg/kg/day of either saline orcG-2-AllylP for 7 days. On day 21 of the experiment the animals weresacrificed and the hippocampal tissue was harvested. Semi-thin sectionsof the tissue were fixed with OsO₄ and embedded in resin. CA1 stratumoriens and CA3 sections were then sliced into ultra-thin, 80 nm slicesand stained with uranyl acetate and lead citrate. Approximately 30synapses per animal were analysed, synapse type classified and vesicledensity was measured using AnalySIS®.

Transmission electron microscopy was used to count the total number ofvesicles on the slides. The average density was calculated by measuringthe total area (using AxioVision software) and using the number ofvesicles. We followed the protocol in Yoshida et al. 97, Journal ofNeurochemistry to calculate the vesicle density in a 200 nm×200 nmsquare apposing the post-synaptic density (PSD).

Results

FIG. 6 is a graph showing the effect of cG-2-AllylP on the trend toincrease the density of pre-synaptic stain in CA3 region of thehippocampus at day 24 post-treatment.

FIG. 7 is a graph showing the effect of cG-2-AllylP on the trend toincrease the density of the pre-synaptic stain in the stratum oriens ofthe CA1 region on day 24 post-treatment.

FIG. 8 is a graph showing the effect of cG-2-AllylP to increase thedensity of the pre-synaptic stain in the stratum radiatum of the CA1region on day 24 post-treatment.

The number of pre-synaptic vesicles in the CA1 and C3 subregions of thehippocampus was increased after cG-2-AllylP treatment (3 mg/kg/day×7days, s.c.) compared to the vehicle treated animals at 21 days after thetreatment (FIG. 11).

We conclude from these studies that scopolamine treatment can decreasecognitive function in animals, and that these changes can mimiccognitive impairment in human beings with one or more of a variety ofneurological conditions. Additionally, we conclude that cG-2-AllylP canimprove cognitive function in scopolamine-treated animals and in animalswith normal aging-related cognitive impairment. Further, we concludethat cG-2-AllylP can increase synaptogenesis, increase AMPA receptors,increase neural plasticity, can stabilize synaptic modifications and canincrease novelty recognition.

These studies therefore support the use of cG-2-AllylP as an effectivepharmacological agent to treat a variety of cognitive impairments inanimals including humans suffering from Alzheimer's disease, Parkinson'sdisease, and other chronic neural disorders, as well as cognitiveimpairment associated with aging.

Example 8: Effects of Cyclic G-2-AllylP and Cyclic cyclopentyl-G-2MeP onCerebellar Cell Explants

To determine the effects of cG-2-AllylP and cyclic cyclopentyl-G-2-MePon neuronal cells in vitro, a series of studies was carried out usingcerebellar explants from adult rats. In vitro systems are suitable forstudying neuronal proliferation, neurite growth, formation of nervebundles, and effects of toxins on neural cells, effects that paralleleffects observed in vivo. Thus, results of studies using in vitrocerebellar explants are predictive of effects of interventions in vivo.

In a first series of studies, effects of glutamate on cerebellarexplants were determined. At physiological concentrations, glutamate isa neurotransmitter in the CNS of mammals, including humans. However, atsufficiently high concentrations, glutamate is neurotoxic, resulting inneuronal cell death. Because glutamate is a naturally occurringneurotransmitter in the CNS of mammals, including humans, and becauseglutamate neurotoxicity is recognized in the art as reflective ofneurotoxicity in general, and including cell death and degeneration, itis a valuable tool useful for identifying and characterizing agentseffective in treatment of neurodegeneration and neural cell death.

Materials and Methods

Cover slips were placed into a large Petri dish and washed in 70%alcohol for 5 minutes, then washed with Millipore H₂O. The cover slipswere air dried, and coated with Poly-D-Lysine (1 mg/ml stock solution inPBS, 90-100 μl) for 2 hours at 34° C.

Extraction of Cerebellar Tissue

Postnatal day 8 Wistar rats were used for the study. The rats weresacrificed and placed in ice for 1 minute, decapitated and thecerebellum removed and placed on ice. Cerebellum tissue was placed in 1ml of 0.65% glucose-supplemented PBS (10 μl 65% stock D (+)glucose/1 mlPBS) in a large Petri dish, chopped up into smaller sections andtriturated with a 1 ml insulin syringe via a 23 G (0.4 mm) needle, andthen squirted back into the glucose solution in the large Petri dish.The tissue was sieved through (125 μm pore size gauze) and centrifuged(2 minutes at 60 g) twice to exchange the medium into serum-freeBSA-supplemented START V medium (Biochrom, Germany). The secondcentrifugation step was done with 1 ml of START V medium. Themicroexplants were reconstituted into 500 μl of START V medium and puton ice.

Cultivation of Cerebellar Ceils

Two hours after PDL-coating, the slides were washed with Millipore H₂Oand air dried. Each slide was placed into a small Petri dish (diameter:35 mm) and 40 μl of START V/cell suspension was added. The tissue wasincubated for 2 hours at 34° C. (settlement period). START V-medium (1ml) was then added to the Petri dish and cultivated at 34° C. in thepresence of 5% CO₂ in air at 100% humidity for 48 hours.

Drug Application

For the study, certain explant cultures were exposed to vehicle (PBS)only. In the first study (Study 1) 10 μl of toxin 1 (L-glutamate—100 mMin Millipore water; final concentration; 1 mM) and 10 μl of toxin 2(3-nitropropionic acid—50 mM—pH 7—in Millipore water, finalconcentration: 0.5 mM) was applied simultaneously with the drug to betested (10 mM stock solution prepared in PBS and diluted to finalconcentrations between 1-100 nM). In each case, the drugs were left incontact with the explants for the duration of the study.

Methods for Determining Drug Effects

After explants were exposed to drugs for the study period, cells werethen rinsed in PBS and then fixed in increasing concentrations ofparaformaldehyde (500 μl of 0.4% PFA was applied; then 1.2% PFA; then 3%PFA and finally 4% PFA (each fixation step: 2-3 minutes). Finally, themicroexplants were rinsed in PBS.

Neurons in the explants were then evaluated for morphology (presence ofneurites) and counted as live cells per microscopic field. Four fieldsdisplaying highest cell density were counted per cover slip and the datapresented as mean±standard error of the mean (SEM); n=4 each.Statistical significance was evaluated by using the non-paired Student'st-test.

Results

Cyclic G-2-AllylP

The results of the study are shown in FIG. 12. Glutamate treatment (1mM; filled bar) resulted in about an 85% loss of cerebellar neuronshaving neurites compared to vehicle-treated controls (open bar). Incontrast, cG-2-AllylP significantly increased the numbers of cellshaving neurites in a dose-dependent manner when administeredsimultaneously with glutamate (shaded bars). Treatment with low doses ofcG-2-AllylP (100 pm to 10 nm) showed a significant decrease inglutamate-induced neurotoxicity.

Cyclic Cyclopentyl-G-2-MeP

The results of the study are shown in FIG. 13. Cyclic cyclopentyl-G-2MePsignificantly increased the number of cells having neurites whensimultaneously administered with glutamate (light shaded bars).Treatment with low doses of cyclic cyclopentyl-G-2MeP showed asignificant decrease in glutamate-induced neurotoxicity.

Conclusions

Both cG-2-AllylP and cyclic cyclopentyl-G-2-MeP independently decreasedor prevented glutamate-induced neurotoxicity, indicating that both drugsare neuroprotective and can be used to inhibit neuronal degeneration orcell death.

Example 9: Effects of cG-2-AllylP on Hypoxic-Ischemic Injury I

Materials and Methods

To determine whether cG-2-AllylP might prevent neuronal injury inresponse to stroke, cardiac arterial bypass graft surgery (CABG) orother hypoxic insults, a series of studies were carried out in rats thathad been exposed to hypoxic-ischemic injury (HI).

Adult rats (Wistar, 280-310 g, male) were used. The modified Levinemodel preparation and experimental procedures were used (Rice et al,1981, Ann. Neurol. :9: 131-141; Guan et al J., 1993, Cereb. Blood FlowMetab.: 13(4): 609-16). These procedures in brief, consist of an HIinjury induced by unilateral carotid artery ligation followed byinhalational asphyxia in the animals with an implanted lateralventricular cannula. A guide cannula was stereotaxically placed on thetop of the dura 1.3 mm to the right of the mid-line and 7.3 mm anteriorto the interaural zero plane under halothane anaesthesia. The rightcarotid artery was double ligated two days after the cannulation. After1 hour recovery from the anaesthesia, each of the rats were placed in anincubator where the humidity (90±5%) and temperature (31°±0.5° C.) werecontrolled for another hour, then exposed to hypoxia (6% oxygen) for 10min. The animals were kept in the incubator for an additional 2 hoursbefore treatment.

Nine pairs of rats were treated intracerebral ventricularly (icv) witheither cG-2-AllylP (2 ng) or its vehicle (normal saline) 2 hours afterhypoxic-ischemic insult. Rats in each group were simultaneously infusedwith cG-2-AllylP or its vehicle under light anaesthesia (1.3% halothane)2 hours after the insult. A total volume of 20 μl was infused (icv) over20 minutes by a micro-infusion pump.

Histological examination was performed on rats 5 days after thehypoxic-ischemic injury. The rats were killed with an overdose of sodiumpentobarbital and were perfused transcardially with normal salinefollowed by 10% formalin. The brains were kept in the same fixative fora minimum of 2 days before being processed using a standard paraffinimbedding procedure.

Coronal sections 8 μm in thickness were cut from the striatum, cerebralcortex and hippocampus and were stained with thionin and acid fuchsin.The histological outcome was assessed at three levels: (1) the mid levelof the striatum, (2) where the completed hippocampus first appeared and(3) the level where the ventral horn of the hippocampus just appears.The severity of tissue damage was scored in the striatum, cortex and theCA1-2, CA3, CA4 and dentate gyrus of the hippocampus. Tissue damage wasidentified as neuronal loss (acidophilic (red) cytoplasm and contractednuclei), pan-necrosis and cellular reactions. Tissue damage was scoredusing the following scoring system: 0: tissue showed no tissue damage,1: <3% tissue was damaged, 2: <50% tissue was damaged, 3: >50% tissuewas damaged and 4: >95% tissue was damaged.

Results and Conclusion

The results of this study are shown in FIG. 14. FIG. 14 shows thathypoxic-ischemic injury (left bars of each set) resulted in significantdamage scores in each of the areas of the brain studied. FIG. 14 alsoshows that central administration of a relatively low dose ofcG-2-AllylP (right bars of each set; 2 ng) significantly reduced thetissue damage in each brain region examined compared to the vehicletreated group (p<0.001).

It can be seen that cG-2-AllylP can be neuroprotective against neuraldamage caused by hypoxic-ischemic injury, even when administered afterhypoxic-ischemic injury. This surprising finding indicates thatcG-2-AllylP is a useful agent to treat a variety of conditionscharacterized by neural degeneration or cell death.

Example 10: Effects of cG-2-AllylP on Hypoxic-Ischemic Injury II

Materials and Methods

Materials and methods described in Example 9 were used and the number oftreatment groups was increased. Rats were divided into 5 treatmentgroups treated intracerebral ventricularly (icv) with one of 4 doses ofcG-2-AllylP or with its vehicle (normal saline) 2 hours afterhypoxic-ischemic insult (1: n=10, 2 ng; 2: n=9, 4 ng; 3: n=9, 20 ng; 4:n=10, 100 ng; and 5: n=9, vehicle).

Results

FIG. 15 shows hypoxia alone (vehicle) produces neuronal damage scores inall areas of the brain studied. In animals treated with cG-2-AllylP,hypoxia had less effect, even though the agent was administered afterthe hypoxic/ischemic injury. The neuroprotective effect was observed forall doses of cG-2-AllylP, except for the highest dose (100 ng)administered to the striatum. However, in all other sites and with allother doses, cG-2-AllylP lessened the neural damage effects ofhypoxia/ischemia. Moreover, cG-2-AllylP had an increased efficacy inbrain regions that experienced progressive injury associated withdelayed cell death, such as that associated with apoptosis. In brainregions such as the dentate gyrus and the cerebral cortex, that are moreresistant to HI injury, the progression of injury is known to be slowerand more severe than in the brain regions that are more sensitive to HIinjury such as the striatum and the CA1-2, CA3 and CA4 sub-regions ofthe hippocampus. This result shows that cG-2-AllylP can be beneficial intreatment of chronic neurological disorders.

Example 11: Effects of cG-2-AllylP in Fragile X Syndrome I GeneralMethods

Experiments were conducted in accordance with the United Kingdom Animals(Scientific Procedures) Act of 1986. Fmr1-K02 mice and wildtype (WT)littermates were generated on a C57BL/6J background and repeatedlybackcrossed onto a C57BL/6J background for more than eight generationsand send to Chile by the Jackson's laboratory. Mice were grouping housed(4-6 per cage) and all animals were provided with ad libitum food andwater unless otherwise stated. Mice were maintained on a 12 h light/darkcycle (lights off 19:00 to 7:00) in a temperature-controlled environment(21±1° C.).

Tasks were performed in the order described with no more than one taskperformed per day. All experiments were blind to the researcherperforming the tests and the person injecting the mice.

Parametric data were analysed using two-way ANOVAs (genotype and sex asbetween-subject factors). Where data violated assumptions of normalityor equality of variance, transformations (log 10 or square root) wereutilised. For repeated measures ANOVAs, homogeneity of variance wastested using Mauchly's test of sphericity, and where this was violated,Huyn-Feldt corrections were used. Non-parametric data were analysedusing Mann-Whitney U tests. A p-value <0.05 was considered statisticallysignificant throughout.

No instances of toxicity have been observed. Animals were inspected fordifferences in coat appearance, whether any piloerection is present, eyecondition (runny eyes or porphyria, ptosis) gait appearance, tremor,tail tone, reactivity to handling, etc.

Example 12: Effects of cG-2-AllylP on Hippocampal Neurons from Animalswith Fragile X Syndrome

To determine whether cG-2-AllylP can affect neurons, we carried out aseries of studies on neurons in vitro from wild-type mice orfmr1-knockout mice.

Methods

Hippocampal cell cultures were prepared from wild type and fmr1-knockoutfetal mice (14-16 days of gestation). Briefly, mice were kill bycervical dislocation under chloroform anesthesia, and dissociatedhippocampal cells were plated in 13 mm multiwell vessels (FalconPrimaria). A plating medium of MEM-Eagle's salts (supplied glutaminefree) supplemented with 10% fetal bovine serum was used. Cultures werekept at 37° C. in a humidified 3% CO₂ atmosphere. After 3d in vitro,green fluorescent protein (GFP) was added to monitor dendritic spinemorphogenesis during time-course of culture (Ethell and Yamaguchi, 1999;Ethell et al., 2001, Henkemeyer et al., 2003). The dendritic spinesusually formed between 7 and 14 days in vitro (DVI). By 14 DIV mostdendritic protrusions were spines. The mean (±SD) spine density wasmeasured as number of spines per micrometer and experiments were run intriplicate.

Results

Results of these studies is shown in FIGS. 16A-16D. FIG. 16A depicts apartition culture chamber for measurement of neuronal morphology. FIG.16B shows a photomicrograph of hippocampal neurons treated withcG-2-AllylP (“NNZ 2591”) at a concentration of 0.5 nM. We observed nostatistically significant change (mean±SD, of n=3 independentexperiments: 0.36±0.02).

In contrast, FIG. 16C shows a photomicrograph of cultured hippocampalneurons treated with cG-2-AllylP at a concentration of 5 nM (0.25±0.03).We observed a statistically significant effects when compared to controlcultures (0.26±0.04).

FIG. 16D shows a photomicrograph of cultured hippocampal neurons treatedwith cG-2-AllylP. A significant effect on spine reduction was observedin cultures treated with cG-2-AllylP at a concentration of 50 nM(0.27±0.10). We observed no statistically significant difference fromwild-type animals (0.26±0.05).

We conclude from these studies that cG-2-AllylP reduces dendritic spinesin vitro, and this result indicates that cG-2-AllylP can improveneurological development and function in vivo, and therefore can beuseful in treating Fragile X Syndrome in mice. Because the murine modelfor Fragile X Syndrome has the same genetic mutation found in humanbeings with Fragile X Syndrome, cG-2-AllylP can be effective in treatinghuman beings with Fragile X Syndrome.

Example 13: Effects of cG-2-AllylP on Behavior in Animals With Fragile XSyndrome I: Anxiety and Memory

To determine whether cG-2-AllylP has a beneficial effect in animals withFragile X Syndrome, we carried out a series of studies on memory orhabituation in vivo in wild-type and fmr1-knockout animals.

Methods

Animals

The fmr1-knockout 2 mice (C57BL/6 background) were housed in groups ofthe same genotype in a temperature and humidity controlled room with a12-h light-dark cycle (lights on 7 am to 7 μm). Testing was conductedduring the light phase. Food and water were available ad libitum.Testing was conducted on fmr1-knockout mice and their wild-typelittermates. Experiments were conducted in line with the requirements ofthe UK Animals (Scientific Procedures) Act, 1986.

Studied Groups (n=10 each) were created according to the following.

1. fmr1-knockout (KO) treated with Vehicle

2. Wild type (Wt) treated with Vehicle

3. fmr1-knockout (KO) treated with cG-2-AllylP (“NNZ-2591”)

4. Wild-type (Wt) treated with cG-2-AllylP.

Open Field Test for Anxiety

The Open Field (OF) test is a combined test that is used to determineanxiety/hyperactivity, and for habituation to a novel environment, oneof the most elementary forms of learning, in which decreased explorationas a function of repeated exposure to the same environment is taken asan index of memory. This is normally studied in two sessions of exposureto the open field, a 10-min and a 24 hr habituation session.

FIG. 17 depicts a photograph of the device used for these studies. Theopen field is an exposed space in which movement can be tracked.

The device used for this study is a grey PVC enclosed arena 50×30 cmdivided into 10 cm squares. Mice are brought to the experimental room5-20 min before testing. A mouse is placed into a corner square facingthe corner and observed for 3 min. The number of squares entered (wholebody) and rears (both front paws off the ground, but not as part ofgrooming) are counted. The latency to the first rear is also noted. Themovement of the mouse around the field was recorded with a videotracking device for 300s (vNT4.0, Viewpoint). The latency for the mouseto enter the brightest, central part of the field total time spent inthis central region, and total activity (in terms of path length incentimeters), were recorded.

The open field (OF) test is a test used to characterize explorativebehavior, anxiety, and or hyperactivity in animals habituated to dailyhandling under novel and familiar conditions. During exposure to theopen field mice will habituate to the environment and thus explore less,decreasing the amount movement they show over time.

In the present experiment, we recorded movement and rearing during aninitial exposure (T1), during a second exposure after 10 minutes (T2)and during a third exposure after 24 hours (T3). Failures to reducelocomotion or rearing at 10 minutes and 24 hours indicate deficits inshort and long term memory, respectively.

Results

cG-2-AllylP Decreases Anxiety in Animals with Fragile X Syndrome

FIG. 18 shows graphs of results of the OF test in which the number ofsquares entered (vertical axis) is plotted for each of the treatmentgroups. Vehicle-treated wild-type animals (left bar) travelled a totaldistance of about 80 squares during the T1 test period. Similarly,cG-2-AllylP (“NNZ 2591”)-treated wild-type animals (third bar from left)entered about the same number of squares during this test period.

In contrast, vehicle-treated fmr1-knockout animals (second bar fromleft) entered more squares during the same time period (p<0.001). Themagnitude of this effect was statistically significant and substantial,with these animals entering about 150 squares during the test period T1.However, we unexpectedly found that cG-2-AllylP (“NNZ 2591”)significantly reduced the exploratory behavior of fmr1-knockout animals(right bar), with results comparable to those seen in vehicle-treatedand cG-2-AllylP-treated wild-type animals (third bar from left).

We conclude from this result that cG-2-AllylP decreases anxiety infmr1-knockout animals. Because the fmr1-knockout mice used in this studyhave the same genetic mutation as human beings with Fragile X Syndrome,we conclude that cG-2-AllylP can decrease anxiety in human beings withFragile X Syndrome.

cG-2-AllylP Improves Short-Term Memory In Animals with Fragile XSyndrome

FIG. 19 shows graphs of results of the OF test at time period T2 (10minutes), in which the number of squares entered (vertical axis) isplotted for each of the treatment groups. As with the results at T1(FIG. 18), the vehicle-treated wild-type animals (left bar) and thecG-2-AllylP (“NNZ 2591”)-treated wild-type animals (third bar from theleft) demonstrated normal exploratory behavior, each group enteringabout 45 squares during time period T2. The distance travelled in the T2test period was less than the distance travelled during T1, indicatingthat the animals had become at least partially habituated to the OF testby this time.

Vehicle-treated fmr1-knockout animals (second bar from the left) showedsubstantially more exploratory behavior than either of the first twogroups of animals (p<0.001). In fact, the magnitude of the increase wasabout 2-fold, to about 100 squares.

In contrast, we unexpectedly found that cG-2-AllylP decreased theexploratory behavior of fmr1-knockout animals (right bar), and in fact,normalized the magnitude of their behavior to that of the wild-typeanimals.

We conclude from these results that: (1) Fragile X Syndrome in micedecreased short-term memory or habituation, and (2) cG-2-AllylP improvedshort-term memory or habituation in fmr1-knockout animals. Because thefmr1-knockout mice used in this study have the same genetic mutation ashuman beings with Fragile X Syndrome, we conclude that cG-2-AllylP canimprove short-term memory in human beings with Fragile X Syndrome.

cG-2AllylP Improves Long-Term Memory in Animals with Fragile X Syndrome

FIG. 20 shows graphs of results of the OF test at time period T3, inwhich the number of squares entered (vertical axis) is plotted for eachof the treatment groups. As with the results at T1 (FIG. 18) and T2(FIG. 19), the vehicle-treated wild-type animals (left bar) and thecG-2-AllylP (“NNZ 2591”)-treated wild-type animals (third bar from theleft) demonstrated normal exploratory behavior, each group enteringabout 25-30 squares during T3. The distance travelled in the T3 testperiod was less than the distance travelled during T2, and furtherdecreased compared to that observed at T1, indicating that the animalshad become progressively habituated, and had a better long-term memoryto the OF test by time period T3.

In contrast, vehicle-treated fmr1-knockout animals (second bar from theleft) showed substantially more exploratory behavior (p<0.001), than thevehicle-treated or cG-2-AllylP-treated groups. In fact, the magnitude ofthe increase was about 2-fold, to about 100 squares. Interestingly, infmr1-knockout animals, habituation failed to increase with time andexposure to the OF device. The number of squares entered at time periodT3 was similar to that found at time periods T2 or T1.

We unexpectedly found that as with short-term memory, in fmr1-knockoutanimals, cG-2-AllylP substantially decreased (second bar from left), andin fact, normalized exploratory behavior at T3 compared tovehicle-treated Fmr1-knockout animals, indicating that long-term memoryor habituation had returned to normal.

Conclusions

We conclude from these results that: (1) fmr1-knockout mice exhibiteddecreased long-term memory or habituation, and (2) cG-2-AllylP improvedlong-term memory or habituation in fmr1-knockout animals. Because thefmr1-knockout mice used in this study have the same genetic mutation ashuman beings with Fragile X Syndrome, we conclude that cG-2-AllylP cannormalize long-term memory or habituation in human beings with Fragile XSyndrome.

Example 14: Effects of cG-2-AllylP on Behavior in Animals With Fragile XSyndrome II: Hyperactivity

To determine whether cG-2-AllylP has a beneficial effect onhyperactivity in animals with Fragile X Syndrome, we carried out aseries of studies in vivo in wild-type and fmr1-knockout mice.

Methods

Animals

The fmr1-knockout (KO2) mice (C57BL/6 background) were housed in groupsof the same genotype in a temperature and humidity controlled room witha 12-h light-dark cycle (lights on 7 am to 7 μm). Testing was conductedduring the light phase. Food and water were available ad libitum.Testing was conducted on fmr1-knockout mice and their wild-typelittermates. Experiments were conducted according to requirements of theUK Animals (Scientific Procedures) Act, 1986. The animals were dividedinto four groups:

Vehicle-treated wild-type

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Successive Alleys Test

In one study, we used a “Successive Alleys Test” device. This deviceconsists of four successive, linearly connected increasingly anxiogenicalleys. Each succeeding alley was painted a lighter color, had lowerwalls and/or was narrower than the previous alley. Animals were placedat the closed end of alley 1 (A1), facing the end wall. The latency tofirst enter each alley (A2, A3, and/or A4), the amount of time spent ineach alley, and the number of entries into each alley were recordedduring a total test time of 300 seconds.

For a Successive Alleys Test, we measured the number of entries in Alley1 (A1), Alley 2 (A2), Alley 3 (A3), and Alley 4 (A4) for each of the 4groups of animals described above.

Results

FIGS. 21 and 22 depict results of the Successive Alleys Test. FIG. 21depicts a graph of results obtained in vehicle-treated animals. FIG. 22depicts a graph of results obtained in cG-2-AllylP (“NNZ 2591”)-treatedanimals. The number of entries is shown (vertical axis) for each of thetreatment groups and entry into each alley (A1-A4) are shown. In FIG.21, vehicle-treated animals showed exploratory behavior, withfmr1-knockout animals showing a larger number of entries than thewild-type animals. The vehicle-treated wild-type animals entered arms A1(WT-A1) and A2 (WT-A2) about 3-4 times during the test period, and didnot enter arms A3 (WT-A3) or A4 (WT-A4) significantly.

In contrast, vehicle-treated fmr1 knockout mice had a significantlyshorter latency time to enter the first open alley (p<0.001) and spentsignificantly more time in the open alleys (p<0.001). These animalsentered arms A1 (KO-A1) and A2 (KO-A2) about 10 times during the testperiod. The time the fmr1-knockout animals entered arms A1 and A2 morethan twice the number of times as did wild type animals, indicating asignificantly higher level of hyperactivity than the vehicle-treatedwild-type animals. The vehicle-treated fmr1-knockout animals also mademore crossings between alleys into arms A3 (KO-A3) and A4 (KO-A4)(p<0.0001).

We conclude from these results that fmr1-knockout mice exhibited greateranxiety that wild-type animals.

FIG. 22 shows results of the Successive Alleys Test in cG-2-AllylP (“NNZ2591”)-treated animals. cG-2-AllylP-treated wild-type animals enteredarm A1 (WT-A1) about 2 times during the test period, and entered arm A2(WT-A2) about 4 times during the test period. These results arecomparable to the results obtained for vehicle-treated wild-type animalsshown in FIG. 21.

In contrast, cG-2-AllylP-treated fmr1-knockout animals treated withcG-2-AllylP showed a significant reduction in open arm entries (p<0.001)as well as time spent in the center (p<0.005), indicating a reduction inhyperactivity compared to vehicle-treated fmr1-knockout animals. Themagnitude of the effect of cG-2-AllylP was substantial, withcG-2-AllylP-treated fmr1-knockout animals entered arm A1 (KO-A1) about 4times compared to about 8 times for the vehicle treated fmr1-knockoutanimals shown in FIG. 21. Similarly, cG-2-AllylP-treated fmr1-knockoutanimals entered arm A2 (KO-A2) about 4 times compared to about 10 timesfor the vehicle treated fmr1-knockout animals shown in FIG. 21.cG-2-Ally IP-treated fmr1-knockout animals entered arm A3 (KO-A3) about4 times compared to about 9 times for the vehicle treated fmr1-knockoutanimals shown in FIG. 21. Finally, cG-2-AllylP-treated fmr1-knockoutanimals entered arm A4 (KO-A4) about 3 times compared to about 4 timesfor the vehicle treated fmr1-knockout animals shown in FIG. 21.

Conclusions

We conclude from this study that cG-2-AllylP decreased the anxiety infmr1-knockout mice under these conditions. Because the fmr1-knockoutmice used in this study have the same genetic mutation as human beingswith Fragile X Syndrome, we conclude that cG-2-AllylP can be effectivein treating human beings with Fragile X Syndrome.

Elevated Plus Maze

In another study, we used an “Elevated Plus Maze.” The Elevated PlusMaze test is one of the most widely used tests for measuringanxiety-like hyperactivity in mice. The test is based on the naturalaversion of mice for open and elevated areas, as well as on theirnatural spontaneous exploratory behavior in novel environments. FIG. 23depicts a photograph of a device used in this study. The apparatusconsists of two open arms and two closed arms, crossed in the middleperpendicularly to each other, and a center area. The open arms are moreexposed and therefore create more anxiety in the mice. Mice thereforespend more time in the closed arms and visit them more frequently. Micewere given access to all of the arms and were allowed to move freelybetween them. The number of entries into the open arms, the time spentin the open arms, and time spent in the center were used as indices ofopen space-induced anxiety.

Time Spent in the Closed Arm

FIG. 24 shows results of studies using the Elevated Plus Maze on theamount of time spent in the closed arm (vertical axis) of the device foreach of four groups of animals tested.

As shown in FIG. 24, vehicle-treated wild-type mice (left bar) spentabout 240 seconds in the closed arm. cG-2-AllylP (“NNZ 2591”)-treatedwild-type mice (third bar from left) spent about the same amount of timein the closed arm.

In contrast, vehicle-treated fmr1-knockout animals (second bar fromleft0 spent significantly less time in the closed arm (about 160seconds; p<0.001), indicating a state of hyperactivity in thefmr1-knockout animals. cG-2-AllylP normalized the time spent in theclosed arm in fmr1-knockout animals (right bar). This hyperactivity wassignificantly reduced in the cG-2-AllylP-treated fmr1-knockout animalscompared to the vehicle-treated wild-type and the cG-2-AllylP-treatedwild-type animals. A statistically significant difference was observedbetween the vehicle-treated fmr1-knockout group and the cG-2-AllylPtreated fmr1-knockout group (p<0.001).

We conclude from this study that cG-2-AllylP decreased the hyperactivityof fmr1-knockout mice under these conditions. Because the fmr1-knockoutmice used in this study have the same genetic mutation as human beingswith Fragile X Syndrome, we conclude that cG-2-AllylP can be effectivein treating human beings with Fragile X Syndrome.

Elevated Plus Maze Time Spent In the Open Arm

FIG. 25 depicts graphs of results of this study, in which the time spentin the open arm (vertical axis) is shown for each of the 4 groups ofanimals tested.

The vehicle-treated fmr1-knockout animals (second bar from left) spent asignificantly longer time in the open arms (p<0.001) compared to theirvehicle-treated wild-type littermates (left bar). In contrast,cG-2-AllylP (“NNZ 2591”) normalized the time spend in the open arms bythe fmr1-knockout animals. The time spent in the open arm by thecG-2-AllylP treated fmr1-knockout animals mice did not significantlydiffer from that observed for the vehicle-treated wild type mice (leftbar), or cG-2-AllylP treated wild-type animals (third bar from left).

We conclude from this study that: (1) fmr1-knockout animals exhibitedmore hyperactivity behavior than wild-type animals, and (2) cG-2-AllylPnormalized the hyperactivity. Because the fmr1-knockout mice used inthis study have the same genetic mutation as human beings with fragile XSyndrome, we conclude that cG-2-AllylP can be effective in treatinghuman beings with fragile X Syndrome.

Elevated Plus Maze Time in the Center

FIG. 26 depicts graphs of the time spent in the center of the device(vertical axis), for each of the 4 groups of animals tested. The timespent in the center of the Elevated Plus Maze is recognized in the artas a measure of hyperactivity.

Vehicle-treated wild-type mice (left bar) spent about 30 seconds in thecenter of the maze. cG-2-AllylP (“NNZ 2591”)-treated wild-type mice(third bar from left) spent slightly less time in the center than didvehicle-treated wild-type animals. In contrast, vehicle-treatedfmr1-knockout mice spent significantly more time in the center (secondbar from left; p<0.001) compared to vehicle-treated wild-type animals.

We unexpectedly found that cG-2-AllylP-treated fmr1-knockout mice (rightbar) spent significantly less time than vehicle-treated fmr1-knockoutanimals (second bar from left). We observed statistically significantdifferences in time spent in the center for vehicle-treatedfmr1-knockout animals (second bar from left), compared tovehicle-treated wild-type animals (left bar; p<0.001),cG-2-AllylP-treated wild-type animals (third bar from left; p<0.001), orcG-2-AllylP-treated fmr1-knockout animals (right bar, p<0.001). In fact,we observed no statistically significant differences between the time inthe center spent by either the vehicle-treated wild-type group (leftbar), the cG-2-AllylP-treated wild-type group (third bar from left) orthe cG-2-AllylP-treated fmr1-knockout group (right bar).

We conclude from this study that cG-2-AllylP decreased the hyperactivityof fmr1-knockout mice under these conditions. Because the fmr1-knockoutmice used in this study have the same genetic mutation as human beingswith fragile X Syndrome, we conclude that cG-2-AllylP can be effectivein treating human beings with Fragile X Syndrome.

Example 15: Effects of cG-2-AllylP on Fear Conditioning in Animals withFragile X Syndrome

Fear conditioning to either a cure or a context represents a form ofassociative learning that has been well used in many species. Thedependent measure used in contextual (delay) fear conditioning is afreezing response that takes place following pairing of an unconditionedstimulus (foot shock), with a conditioned stimulus (CS), a particularcontext and/or such a cue. If in a conditioning context one administersa foot shock that is paired with atone, there will be learning not onlyto the tone, but also to the context.

Contextual fear conditioning is a bask conditioning procedure. Itinvolves taking an animal and placing it in a novel environment,providing an aversive stimulus, and then removing it. When the animal isreturned to the same environment, it generally will demonstrate afreezing response if it remembers and associates that environment withthe aversive stimulus. Freezing is a response to fear, which has beendefined as “absence of movement except for respiration.” This freezingbehavior may last from seconds to minutes depending on the strength ofthe aversive stimulus, the number of presentations, and the degree oflearning achieved by the subject.

Methods

Animals

We used either wild-type or fmr1-knockout animals for this study, asdescribed above. The animals were divided into groups as follows.

Vehicle-treated wild-type;

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Apparatus for Assessing Contextual Fear Conditioning

The device used in this study is depicted in FIG. 27. An unconditionedstimulus (mild shock to the feet) was applied, and the conditionedstimulus was a tone applied along with the shock to the feet. Underthese conditions, the animals associate the unconditioned stimulus withboth the conditioned stimulus and the context of the stimuli. Animalswere tested for five (3) minutes.

Results

FIG. 28 depicts a graph of the time spent in “freezing behavior” foreach of the groups of animals tested. Under acute stress conditions ofthis study, vehicle-treated wild-type animals (left bar) spent anaverage of about 30% of the five-minute test period (i.e., about 100seconds). In contrast, vehicle-treated fmr1-knockout animals (second barfrom left) spent substantially less time in freezing behavior (about 18%of the five-minute test period, or about 54 seconds). We conclude thatthe fmr1-knockout animals exhibited less fear than vehicle-treatedwild-type animals.

We unexpectedly found that cG-2-AllylP (“NNZ 2591”) produced asubstantial and statistically significant increase in the time spent infreezing behavior in fmr1-knockout animals (right bar). In fact, thetime spent in freezing behavior observed for cG-2-Allyl P-treatedfmr1-knockout animals was similar to the time spent by thevehicle-treated wild-type animals (left bar) and the cG-2-AllylP=treatedwild-type animals (third bar from left).

Conclusions

We conclude from this study that fmr1-knockout animals exhibited lowerfear conditioning than wild-type animals. This indicates thatfmr1-knockout animals may have a survival disadvantage compared towild-type animals. We also conclude that cG-2-AllylP increased fearconditioning in fmr1-knockout mice under these conditions. This effectmay mitigate the survival disadvantage observed in vehicle-treatedfmr1-knockout animals. Because the fmr1-knockout mice used in this studyhave the same genetic mutation as human beings with Fragile X Syndrome,we conclude that cG-2-AllylP can be effective in treating human beingswith Fragile X Syndrome.

Example 16: Effects of cG-2-AllylP on Marble Burying and NestingBehavior in Animals with Fragile X Syndrome

Mice are a social species, which engage in easily scored socialbehaviors including approaching, following, sniffing, allo-grooming,aggressive encounters, sexual interactions, and parental behaviors,nesting and sleeping in a group huddle, social recognition and socialmemory in mice are evaluated y the amount of time sent sniffing a novelmouse upon repeated exposures, to induce familiarity, and re-instatementof high levels of sniffing when a novel stimulus animal is introduced.

Marble Burying

Mice spontaneously dig in many substrates in the laboratory. Thisbehavior comes from their ancestry in the wild, where they would foragefor seeds, grain, insects, and other food to be found buried in the soilor leaf litter in their natural habitat. It exploits a common naturalrodent behavior, provides quantitative data under controlled laboratoryconditions, and has proved extremely sensitive to prion disease. FragileX Syndrome, and brain lesions. Deterioration in the ability to perform“Activities of daily living” (ADL) is an early sign of Alzheimer'sdisease (AD), and cognitive decline (Deacon, 2012).

To study the effects of cG-2-AllylP on marble burying behavior, weearned out a series of studies on wild-type mice and fmr1-knockout mice.

Methods

Animals

We used either wild-type or fmr1-knockout animals for this study, asdescribed above. The animals were divided into groups as follows.

Vehicle-treated wild-type;

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Mice were placed in an enclosed environment with a bed of wood shavings.Ten (10) marbles were introduced into the environment, and the number ofmarbles buried was determined by visual observation(median±interquartile range (IQR)).

Results

We observed that vehicle-treated wild-type mice and cG-2-AllylP-treatedwild-type mice buried 10 out of 10 (100%) of the marbles presented. Incontrast, fmr1-knockout animals buried significantly fewer marbles(average of 3 of 10 or 30%; (p<0.001) than vehicle-treated wild-typeanimals.

We unexpectedly found that cG-2-AllylP (“NNZ 2591”)-treatedfmr1-knockout animals buried a median of 8 of 10 marbles (80%). In fact,cG-2-AllylP normalized the number of marbles buried to levels verysimilar to those found for vehicle-treated wild-type mice, orcG-2-AllylP-treated wild-type mice.

Conclusions

We conclude from this study that fmr1-knockout mice buried fewer marblesthan wild-type animals, and that cG-2-AllylP rescued this decrease, andin fact, normalized the behavior. Because the fmr1-knockout mice used inthis study have the same genetic mutation as human beings with Fragile XSyndrome, we conclude that cG-2-AllylP can be effective in treatinghuman beings with Fragile X Syndrome.

Nesting Behavior

Males and female mice make nests and perform this test equally, aspurposes include thermoregulation as well as being associated withreproduction. This test is also used as an indicator of hippocampuslesion and dysfunction.

Methods

Animals

We used either wild-type or fmr1-knockout animals for this study, asdescribed above.

The animals were divided into groups as follows.

Vehicle-treated wild-type;

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Test for Nesting Behavior

Mice typically use certain materials with which to make a nest. In thisstudy, we introduced cotton “nestlets” into the nesting cages. Mice canthen tear up and use the cotton of the nestlet and use it to make a nextin the bedding of the nesting cages. Nesting is a typical behavior inmice, and represents an aspect of routine, daily living. This test istherefore reflective of daily living tasks carried out by human beings.Changes in nesting behavior in fmr1-knockout mice are thereforepredictive of the behavior of human beings with Fragile X Syndrome.Further, effects of drugs on fmr1-knockout mice are reasonablypredictive of effects of drugs on human beings with Fragile X Syndrome.

Mice were placed individually into nesting cages about one hour beforethe dark phase of the light-dark cycle, and the results were assessedthe next morning. The appearance of the nestlets were evaluatedaccording to a 5-point scale as described below, and the amount ofuntorn nestlet material was also weighed.

1. The nestlet was largely untouched (>90% intact). FIG. 20A depicts aphotograph of a nestlet having a score of 1.

2. The nestlet was partially torn up (30-90% remaining intact). FIG. 20Bdepicts a photograph of a nestlet having a score of 2.

3. The nestlet was mostly shredded but often there is no identifiablenest site: <30% of the nestlet remains intact but <90% is within aquarter of the cage floor area, i.e. the cotton was not gathered into anest but spread around the cage. The material may sometimes be in abroadly defined nest area but the critical definition is that 30-90% hasbeen shredded. FIG. 29C depicts a photograph of a nestlet having a scoreof 3.

An identifiable, but flat nest: >90% of the nestlet was torn up, thematerial was gathered into a nest within a quarter of the cage floorarea, but the nest is flat, with walls higher than mouse body height(curled up on its side) on less than 30% of its circumference. FIG. 29Ddepicts a photograph of a nestlet having a score of 4, with a mouse ontop of the nestlet.

3. A near perfect nest: >90% of the nestlet was tom up, the nest is acrater, with walls higher than mouse body height on more than 30% of itscircumference. FIG. 29E depicts a photograph of a nestlet having a scoreof 3, with a mouse on top of the nestlet.

Results and Conclusions

We found that vehicle-treated wild-type C37BL/6 mice scored betweenabout 4-3 on nest construction. In contrast, for the vehicle-treatedfmr1-knockout mice, the median score was around 1-2.

We unexpectedly found that cG-2-AllylP (“NNZ 2391”) treatment increasedthe nesting score to about 4-3 in fmr1-knockout mice.

We conclude that fmr1-knockout mice demonstrated a deficit in nextbuilding, and that cG-2-AllylP at least partially reversed this deficit.Because the fmr1-knockout mice used in this study have the same geneticmutation as human beings with Fragile X Syndrome, we conclude thatcG-2-AllylP can be effective in treating human beings with Fragile XSyndrome.

Example 17: Sociability: Social Recognition, Preference for SocialNovelty

Mice are a social species, which engage in easily scored socialbehaviors including approaching, following, sniffing, allogrooming,aggressive encounters, sexual interactions, parental behaviors, nestingand sleeping in a group huddle. To study sociability, we carried out aseries of studies in wild-type mice, and mice with the fmr1-knockoutmutation.

Methods

Animals

We used either wild-type or fmr1-knockout animals for this study, asdescribed above.

The animals were divided into groups as follows.

Vehicle-treated wild-type;

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Test Procedures

Social recognition and social memory in mice were evaluated by theamount of time spent sniffing a novel mouse upon repeated exposures, toinduce familiarity, and reinstatement of high levels of sniffing when anovel stimulus animal is introduced. We measured the number of bouts ofsniffing in each of the groups of animals.

Results and Conclusions

FIG. 30 depicts a graph of the duration of bouts of sniffing (verticalaxis) for the different groups of mice studied. We found thatvehicle-treated wild-type animals (left bar) exhibited bouts of sniffingof about 23 during the test period. In contrast, fmr1-knockout animals(second bar from left) exhibited less sniffing behavior (about 6).

We surprisingly found, however, that in cG-2-AllylP (“NNZ 2591”)-treatedfmr1-knockout animals (right bar), the amount of sniffing behaviorsignificantly increased to about 21 (p<0.001). In fact, cG-2-AllylPincreased sniffing behavior in fmr1-knockout animals to about the samelevels as in vehicle-treated wild-type (left bar) or cG-2-AllylP-treatedanimals (third bar from left).

We conclude that fmr1-knockout animals exhibited a deficit ofsociability compared to wild-type animals. The results of this areconsistent with the well-known deficits in sociability observed in humanbeings with Fragile X Syndrome. We also conclude that cG-2-AllylPincreased sociability observed for fmr1-knockout mice, and normalizedthe behavior.

Because the fmr1-knockout mice used in this study have the same geneticmutation as human beings with Fragile X Syndrome, we conclude thatcG-2-AllylP can be effective in improving social interactions in humanbeings with Fragile X Syndrome.

Example 18: cG-2-AllylP Normalizes Overexpression of pERK and pAKT inAnimals with Fragile X Syndrome

Neurons are critically influenced by Fragile X Mental RetardationProtein, which regulates local dendritic translation throughphosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (mTOR)and Ras-ERK signalling cascades and implicated in the mGluR5 signallingcascade. Over-activation of the intracellular signalling molecules, ERKand Akt play a crucial role in synaptic plasticity. Levels of expressionof these proteins is a feature of the cellular pathology of Fragile XSyndrome and is believed to contribute directly to the neurobehaviouralphenotype of FXS. ERK is a classical MAPK signal transduction protein,responsible for growth factor transduction, proliferation, cytokineresponse to stress and apoptosis. Akt is a key component in thePI3K/Akt/mTOR signalling pathway and regulates cellular survival andmetabolism by binding and regulating many downstream effectors, such asNuclear Factor-κB (NfκB) and Bcl-2 family proteins. Excess activation(phosphorylation) of these proteins has been implicated in autismspectrum disorders.

Methods

Animals

We used either wild-type or fmr1-knockout animals for this study, asdescribed above.

The animals were divided into groups (n=4 animals per group) as follows.

Vehicle-treated wild-type;

Vehicle-treated fmr1-knockout;

cG-2-AllylP-treated wild-type; and

cG-2-AllylP-treated fmr1-knockout.

Biochemical Tests

Phosphorylation levels of ERK1/2 and Akt of full brain lysates wereevaluated using Western blots. The mice were sacrificed and the brainsremoved 12 days after the last injection, and immediately after the lastbehavioral test. Other studies measured pERK and pAkt in bloodlymphocytes. The results of Western analysis were normalized to theamount of GAPDH protein seen in each blot.

Results

Phosphorylated ERK

FIG. 31 depicts graphs of results of the study on brains from the mice.Vehicle-treated wild-type mice had an average of about 0.9 (UA units)(left bar). In contrast, fmr1-knockout animals (second bar from left)had an increased level of phosphorylation ERK to about 1.3 (UA units)(p<0.03). Treatment of fmr1-knockout animals with c-2-AllylP (“NNZ2591”) (third bar from left) significantly reduced ERK phosphorylation(p<0.03) compared to vehicle-treated fmr1-knockout animals. The levelsof pERK observed after treatment of fmr1-knockout animals withcG-2-AllylP were very similar to those of vehicle-treated wild-typeanimals (left bar) or cG-2-AllylP treated wild-type animals (third barfrom left).

Similar results were found for pERK in lymphocytes isolated from thegroups of animals.

Phosphorylated AKT

We observed a similar pattern in phosphorylated AKT as we did for ERK inbrains of the animals. FIG. 32 depicts graphs of results of this study.Vehicle-treated wild-type mice had an average of about 0.9 (UA units)(left bar). In contrast, fmr1-knockout animals (second bar from left)had an increased level of phosphorylation AKT of about 1.3 (UA units)(p<0.05). Treatment of fmr1-knockout animals with c-2-AllylP (“NNZ2591”) (third bar from left) significantly reduced ERK phosphorylationto about 0.9 (UA units (p<0.05) compared to vehicle-treatedfmr1-knockout animals. The levels of pAKT observed after treatment offmr1-knockout animals with cG-2-AllylP were very similar to those ofvehicle-treated wild-type animals (left bar) or cG-2-AllylP treatedwild-type animals (third bar from left).

Similar results were found for pAkt in lymphocytes isolated from thegroups of animals.

Example 19: Treatment of Rett Syndrome: Effects of cG-2-AllylP onLifespan and Long-Term Potentiation in Rett Syndrome (RTT) Model

To determine whether cG-2-AllylP treatment can impact the developmentand progression of Rett Syndrome in a murine model of the disorder, weuse hemizygous MeCP2(1lox) male mice. The MeCP2 knock-out (MeCP2-KO)mouse system is widely accepted in the art as closely mimicking therange and the severity of physiological and neurological abnormalitiescharacteristic of the human disorder, Rett Syndrome.

All experiments are performed at the University of Texas SouthwesternMedical Center and approved by the University of Texas SouthwesternMedical Center Animal Care and Use Committee or similar approvals byother organizations. cG-2-AllylP was synthesised Albany MolecularResearch Inc. (Albany, N.Y.) and supplied by Neuren PharmaceuticalsLimited.

Methods

Treatment

We treat hemizygous MeCP2(1lox) male mice with 20 mg/kg/day ofcG-2-AllylP or saline, (0.01% BSA, n=15 per group in survival experimentand n=20 in the LTP experiment). The treatments are administeredintraperitoneally from 4 weeks after birth. For the survival experimentsthe treatment is maintained through the course of the experiment. Forthe LTP experiment the mice are treated until week 9 when they are usedfor slice preparation.

Survival

MeCP2 deficient mutant mice develop RTT symptoms at about 4-6 weeks ofage and the between 10-12 weeks (Chen et al., 2001. Nat Genet 27:327-331). We compare the survival of the wild type controls and theMeCP2 deficient animals in vehicle- and cG-2-AllylP-treated groups.Survival is measured weekly from start of treatment (4 weeks) and usedto produce Kaplan-Meier survival curves to show the proportion of micethat survive (y axis) at each weekly interval (x axis).

Long-Term Potentiation (Electrophysiology)

MeCP2 deficient mice have been previously reported to suffer fromfunctional and ultrastructural synaptic dysfunction, significantimpairment of hippocampus-dependent memory and hippocampal long-termpotentiation (LTP) (Moretti et al. The Journal of Neuroscience. 2006.26(1):319-327). To test the effects of the cG-2-AllylP treatment onsynaptic function in the RTT model we compare hippocampal LTP in bothvehicle and cG-2-AllylP-treated animals at 9 weeks of age. To do so, wemeasure the slope of the fEPSP as a % of baseline potential in neuronsin slices of hippocampus from MeCP2 deficient mice treated with eithersaline or cG-2-AllylP.

Results

Results show that cG-2-AllylP treatment increases survival of MeCP2deficient mice. Wild-type mice (top line) are control animals, andtherefore their survival is 100% at each time point. MeCP2 deficientmice treated with saline only the much more rapidly than wild-type mice,such that by about 11 weeks, only some of the MeCP deficient micesurvive. In contrast, however, we find that MeCP2 deficient mice treatedwith cG-2-AllylP survive substantially longer than saline-treated mice.No safety concerns are raised by cG-2-AllylP treatment of mecp2 mice.

These results demonstrate that cG-2-AllylP can substantially increasesurvival of MeCP2 deficient mice. Because MeCP2 deficient mice arepredictive of the pathology and therapeutic efficacy in human beingswith Rett Syndrome, we conclude that cG-2-AllylP can increase life spanof human beings with Rett Syndrome.

Results also show that cG-2-AllylP treatment increases hippocampallong-term potentiation (LTP) as measured by the fEPSP slope in MeCP2deficient animals compared to saline-treated mutant mice. We find thatcG-2-AllylP increases the slope of fESPS in MeCP2 deficient micecompared to animals treated with saline only.

These results demonstrate that cG-2-AllylP can be effective in treatingMeCP2 deficient mice in vivo. Because MeCP2 deficient mice arepredictive of the pathology and therapeutic efficacy in human beingswith Rett Syndrome, we conclude that cG-2-AllylP can be an effectivetherapy for human beings with Rett Syndrome.

Example 20: cG-2-AllylP Improves Dendritic Arborization and IncreasesDendritic Spine Length

We assess the effects of cG-2-AllylP treatment on dendrites. Transgenicmecp2 knockout mice (n=15 to 20) are administered cG-2-AllylPintraperitoneally at a dose of 20 mg/kg once daily. Following sacrificedendritic spine density, spine length and aborization are examined afterGolgi staining after nine weeks, according to Table 5 below.

TABLE 5 Sample Sizes for all Neuron Morphology and Spine Analysis No. ofof MALE neurons or No. of mice dendrites per animal AGE KO- KO-cG-2- KO-KO-cG-2- Analysis (Weeks) vehicle AllylP vehicle AllylP Morphology 9 3 34 4 Spine 9 3 3 10 10 Analysis

Dendritic length is assessed by distance from the soma of representativehippocampal CA1 neurons from 9 week old male mecp2 null mutant micetreated with either saline (3 neurons analysed from 3 separate mice,n=9) or cG-2-AllylP (20 mg/kg i.p. 1/day, from week 4; 3 neurons areanalysed from 3 separate mice, n=9).

We observe that cG-2-AllylP improves dendritic arborization andincreases dendritic spine length. Dendritic length in μm (vertical axis)is plotted against the distance (in μm; horizontal axis) from the somaof the cells. For cells with dendrites close to the so mas, thedendrites are short. However, as the distance from the somas increases,saline-treatment produces dendritic lengths that increase to a maximumat a distance of about 70 μm from the soma and decline at distancesfurther away from the somas. In contrast, treatment with cG-2-AllylP(filled squares) produces longer dendrites over much of the range ofdistances from the somas.

Example 21: Treatment of Rett Syndrome in Mice II: Mice Mating andGenotyping

The MeCP2 germline null allele mice are used (Chen et al., 2001).Genotyping is performed as in Chen et al. (Chen et al., 2001).

cG-2-AllylP Treatment

For the survival measurements, the nocturnal activity analysis and theimmunoblot analysis, cG-2-AllylP supplied by Neuren PharmaceuticalsLimited is administered daily via intra-peritoneal injections (20 mg/kg,vehicle=saline, 0.01% BSA). The treatment starts at PI 5 and ismaintained throughout the course of the experiments. For intracellularphysiology experiments, the mice are injected daily with cG-2-AllylP (20mg/kg body weight, vehicle=saline, 0.01% BSA) for 2 weeks, from P15 toP28-P32 when they are used for acute slice preparation. For opticalimaging experiments, mice are injected with cG-2-AllylP (20 mg/kg bodyweight, vehicle=saline, 0.01% BSA) daily from the day of the lid sutureto the day of imaging.

Slice Physiology Preparation

Coronal sections (300 μm thick) at or near sensorimotor cortex are cutin <4° C. ACSF using a Vibratome. Slices are incubated at 37° C. for 20minutes after slicing, and at room temperature for the remainder of theexperiment. Slices are transferred to a Warner chamber and recordingsare taken from visually identified pyramidal neurons located in layer 3.Artificial cerebral spinal fluid (ACSF) containing 126 mM NaCl, 23 mMNaHCO₃, 1 mM NaHPO₄, 3 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, and 14 mMdextrose, is adjusted to 313-320 mOsm and 7.4 pH, and bubbled with 93%02/3% CO₂. The intracellular pipette solution contains 100 mM potassiumgluconate, 20 mM KCl, 10 mM HEPES, 4 mM MgATP, 0.3 mM NaGTP, and 10 mMNa-phosphocreatine.

Intracellular Whole-Cell Recordings

Borosilicate pipettes (3-3 MΩ, WPI) are pulled using a Sutter P-80puller (Sutter Instruments). Cells are visualized with an Achroplan 40×water-immersion lens with infrared-DIC optics (Zeiss) and detected withan infrared camera (Hamamatsu) projecting to a video monitor.Experiments are driven by custom acquisition and real-time analysissoftware written in Matlab (Mathworks, Natick, Mass.) using a Multiclamp700B amplifier (Axon Instruments) connected to a BNC-2110 connectorblock and M-Series dual-channel acquisition card (National Instruments).Gigaseal and rupture is achieved and whole-cell recordings arecontinuously verified for low levels of leak and series resistance. Foreach recording, a 3 mV test pulse is applied in voltage clamp ˜10 timesto measure input and series resistance. Then in current clamp ˜10 pulses(300 ms, 40-140 pA at 10 pA increments), are applied to quantify evokedfiring rates and cellular excitability. Access resistance, leak, andcellular intrinsic excitability are verified to be consistent acrossgroups. Finally, spontaneous EPSCs under voltage clamp at −60 mV aresampled at 10 kHz and low-pass filtered at 1 kHz. Analysis is performedusing a custom software package written in Matlab, with all eventsdetected according to automated thresholds and blindly verified for eachevent individually by the experimenter.

Golgi Staining

Samples (<1 cm) from P28 mice are fixed in 10% formalin and 3% potassiumbichromate for 24 hours. Tissue is then transferred into 2% silvernitrate for 2 days in the dark at room temperature. Sections from thesesamples are then cut at 30 μm thickness into distilled water. Sectionscorresponding to motor cortex are mounted onto slides, air dried for 10minutes, and then dehydrated through sequential rinses of 93% alcohol,100% alcohol, and xylene, and then sealed with a coverslip. Images reacquired at 10× (whole cell) and 100× (spine imaging) using a ZeissPascal 3 Exciter confocal microscope.

Optical Imaging of Intrinsic Signals

Adult (>P60) wild type (SVEV or BL6) and MeCP2 (+/−) mutant females(BL6) are used for this experiment. The wild type control group iscomposed of both wild type littermates of MeCP2+/− females or wild typeage matched SVEV females. For monocular deprivation, animals areanesthetized with Avertin (0.016 ml/g) and the eyelids of one eye issutured for 4 days. Prior to imaging, the suture is removed and thedeprived eye re-opened. Only animals in which the deprivation suturesare intact and the condition of the deprived eye appears healthy areused for the imaging session. For cG-2-AllylP signaling activation, asolution containing cG-2-AllylP is injected intra-peritoneally (IP)daily for the entire period of deprivation. For the imaging sessionsmice are anesthetized with urethane (1.3 g/kg; 20% of the full dosage isadministered IP each 20-minutes up to the final dosage, 0.02 ml ofcloroprothixene 1% is also injected together with the firstadministration). The skull is exposed and a custom-made plate is gluedon the head to minimize movement. The skull is thinned over VI with adremel drill and covered with an agarose solution in saline (1.3%) and aglass coverslip. During the imaging session, the animal is constantlyoxygenated, its temperature maintained with a heating blanket and theeyes periodically treated with silicone oil; physiological conditionsare constantly monitored. The anesthetized mouse is placed in front of amonitor displaying a periodic stimulus presented to either eye,monocularly; the stimulus consisted of a drifting vertical or horizontalwhite bar of dimensions 9°×72°, drifting at 9 sec/cycle, over auniformly gray background. The skull surface is illuminated with a redlight (630 nm) and the change of luminance is captured by a CCD camera(Cascade 512B, Roper Scientific) at the rate of 15 frames/sec duringeach stimulus session of 23 minutes. A temporal high pass filter (133frames) is employed to remove the slow signal noise, after which thesignal is computer processed in order to extract, at each pixel, thetemporal Fast Fourier Transform (FFT) component corresponding to thestimulus frequency. The FFT amplitude is used to measure the strength ofthe visual evoked response to each eye. The ocular dominance index isderived from each eye's response (R) at each pixel asODI=(Rcontra−Ripsi)/(Rcontra+Ripsi). The binocular zone is defined asthe region activated by the stimulation of the eye ipsilateral to theimaged hemisphere.

Heart Rate Measurements

Real time cardiac pulse rate is measured using a tail clip sensor (MouseOX Oximeter-Oakmont, Pa.). Mice are not anesthetized but physicallyrestrained in a fitted open plastic tube. Prior to the recording sessionthe tube is placed overnight in the cages housing the experimentalanimals to allow habituation. Body temperature is maintained at ˜82-84°F. throughout the recording time. We record 3 trials of 15 minutes foreach mouse, mice are 8 weeks old and treated with vehicle or cG-2-AllylPfrom PIS.

Nocturnal Activity Measurements

Spontaneous motor activity is measured by using an infraredbeam-activated movement-monitoring chamber (Opto-Varimax-MiniA; ColumbusInstruments, Columbus, Ohio). For each experiment, a mouse is placed inthe chamber at least 3 h before recordings started. Movement ismonitored during the normal 12-h dark cycle (7 p.m. to 7 a.m.). One darkcycle per animal per time point is collected.

Results

To test whether cG-2-AllylP treatment will impact the development ofcardinal features of the RTT disease, 2 week old mutant animals aregiven daily intra-peritoneal injections for the course of theirlifespan. Measurements of synaptic physiology, synaptic molecularcomposition, and cortical plasticity are then acquired as detailedbelow, along with health-related measurements such as heart rate,locomotor activity levels, and lifespan.

Effects of cG-2-AllylP on the Synaptic Physiology of MeCP2 Mutant Mice

Recent studies have reported that neurons across multiple brain regionsof MeCP2−/y mice display a profound reduction in spontaneous activity(Chang et al., 2006; Chao et al., 2007; Dani et al., 2005; Nelson etal., 2006) a phenotype that is rescued by over-expression of BDNF (Changet al., 2006). Similarly, acute application of an IGF1 derivative hasbeen shown to elevate evoked excitatory postsynaptic current (EPSC)amplitudes by 40% in rat hippocampal cultures (Ramsey et al., 2005; Xinget al., 2007). To test the efficacy of cG-2-AllylP in rescuing theMeCP2-/y physiological phenotype, we acquire intracellular whole cellrecordings in acute brain slices, measuring excitatory synaptic drive(spontaneous EPSC amplitude and frequency) in layer 5 cortical neurons.Here, EPSCs recorded from −/y animals are significantly reduced inamplitude compared to EPSCs measured in wild-type animals. The trend ispartially reversed in EPSCs recorded from MeCP2−/y animals treated withcG-2-AllylP, which are significantly larger in amplitude than EPSCs fromMeCP2−/y mice treated with vehicle. These differences are also seen whenaveraging across cells. Throughout these measurements, accessresistance, leak, and cellular intrinsic excitability are also verifiedto be consistent across groups. Quantifying EPSC intervals also shows aslight increase in the interval between EPSC events (reduced EPSCfrequency) between wild-type and MeCP2−/y animals (P=0.04,Kolmogorov-Smirnov test). We find that the reduction of excitatorysynaptic drive in cortical cells of MeCP2−/y mice, and its partialrescue following cG-2-AllylP treatment, are due in part to a change inEPSC amplitude as a consequence of a change in the strength of thesynapses mediating excitatory transmission in this region.

cG-2-AllylP Treatment Stimulates Cortical Spine Maturation

We use Golgi staining to label neurons sparsely and distinctly, andapplied high-resolution confocal imaging to measure dendritic spinedensity and morphology in the labelled cells, restricting analysis tolayer 5 pyramidal neurons in sections of motor cortex from criticalperiod mice (P28).

While low-magnification imaging clearly delineates the extent of thedendrites of the pyramidal cells we use higher magnifications to countsynaptic contacts and determine the morphological class of each spine.We classify spines as either large and bulbous (“mushroom”, M), shortand stubby (“stubby”, S), short and thin (“thin”, T) or filopodia (F).Comparing the density of spines per unit branch exhibits a trend ofdecreased spine density in knockout neurons that is largely amelioratedin the knockout with treatment.

We find the potential for deficits in the number and maturational statusof dendritic contacts in the knockout to underpin functional defects inexcitatory transmission, in a manner that can be treated followingadministration of cG-2-AllylP.

Ocular Dominance (OD) Plasticity in Adult MeCP2+/− Mice is Reduced BycG-2-AllylP

Developmental changes in OD plasticity are controlled in part by theactivation of the IGF-1 pathway, and administration of (1-3)IGF-1 canreduce OD plasticity in wild type young mice (Tropea et al., 2006). Wetherefore test if cG-2-AllylP treatment could stabilize the prolonged ODplasticity observed in adult MeCP2 mutants. Female MeCP2+/− mice, agedP60 or more, are monocularly deprived for 4 days and treatedconcurrently with cG-2-AllylP. cG-2-AllylP treatment reduces the ODplasticity in the adult Mecp2+/− mice, indicating that indeedcG-2-AllylP can rapidly induce synapse stabilization or maturation.

Bradycardia in MeCP2−/y Mice is Treated by cG-2-AllylP

In addition to examining the efficacy of cG-2-AllylP in amelioratingneurophysiological symptoms, we seek to characterize its effects on thegeneral health of the organism. Clinical and experimental evidence showsautonomic system dysfunctions such as labile breathing rhythms andreduced baseline cardiac vagal tone in Rett Syndrome patients (Julu etal., 2001). A poor control of the feedback mechanisms that regulateblood pressure homeostasis through the sympathetic system, for examplehyperventilation-induced decrease in heart rate, is common in RettSyndrome patients and can cause life threatening cardiac arrhythmias(Acampa and Guideri, 2006; Julu et al., 2001).

The pathogenesis of the cardiac dysautonomia, although not wellunderstood, suggests that immature neuronal connections in the brainstemcould be the cause. To examine heart rate abnormalities in MeCP2−/y miceand the effect of cG-2-AllylP treatment, we monitor real time cardiacpulse rate in non-anesthetized wild type and MeCP2−/y animals treatedwith vehicle or cG-2-AllylP. Wild type mice exhibit a regulardistribution of heart rate measurements centred near 750 beats perminute. In contrast, MeCP2−/y mice exhibit a more irregular heart ratewith a lower average rate, the occurrence of which is significantlyreduced following treatment with cG-2-AllylP.

cG-2-AllylP Administration Improves Locomotor Activity and Life Span

MeCP2−/y mice develop Rett-like symptoms beginning at 4-6 weeks of agewhen they progressively become lethargic, develop gait ataxia and thebetween 10 and 12 weeks of age (Chen et al., 2001). Baseline locomotoractivity is also recorded in mice after 6 weeks by counting nocturnalinfrared beam crossing events within a caged area. MeCP2 knockout mice(KO) exhibits markedly reduced locomotor activity levels compared towild-type mice (WT), but treatment with cG-2-AllylP (KO-T) elevatesthese levels.

Finally, compared to MeCP2 KO littermates, MeCP2−/y mice treated withcG-2-AllylP also show an increase in life expectancy.

We also measure the effect of cG-2-AllylP treatment on neuron soma sizein the hippocampus. Mice are treated with cG-2-AllylP as described abovefor locomotor activity. Soma size in neurons in the CA3 region of thehippocampus is significantly impaired in MeCP2 KO animals relative towild-type animals. cG-2-AllylP treatment increases average soma size inKO animals, but has little or no effect on soma size in wild typeanimals.

Example 22: Effect of Oral cG-2-AllylP on Survival in Rett Syndrome inMice

Because Rett Syndrome is a chronic, debilitating disorder involving lossof motor skills, it is desirable to treat Rett Syndrome using easilyadministered preparations. To this end, we can take advantage ofunexpectedly beneficial therapeutic and pharmacokinetic properties ofcG-2-AllylP and related compounds (U.S. Pat. Nos. 7,776,876, and8,067,425).

Therefore, we administer cG-2-AllylP orally to MeCP2 deficient mice.Briefly, an aqueous solution or other composition containing apharmaceutically effective amount of cG-2-AllylP (20 or 80 mg/kg peranimal) is administered daily. In control MeCP2 deficient animals, weadminister saline only, and wild-type animals are used to obtainbaseline data similar to the design.

In wild-type animals, survival is defined to be 100% at each time point.In MeCP2 deficient animals, survival is decreased substantially.However, after oral administration of cG-2-AllylP to MeCP2 deficientmice, survival is increased substantially.

Example 23: Effect of cG-2-AllylP on Seizure Activity in Rett Syndromein Mice

Because seizures are a prominent, hazardous and a difficult to treataspect of Rett Syndrome, we determine the effects of cG-2-AllylP onseizure activity in MeCP2 deficient animals.

Electroencephalograph: recordings of wild-type mice and MeCP2 deficientmice treated with either saline or cG-2-AllylP are obtained usingmethods described in U.S. Pat. No. 7,714,020, incorporated fully byreference.

We find that cG-2-AllylP can be effective in decreasing both motorseizures and non-convulsive seizures.

Conclusions

cG-2-AllylP can be an effective therapy for treating human beings withRett Syndrome. Moreover, because cG-2-AllylP has unexpectedly longerhalf life than a naturally occurring compound ((1-3) IGF-1;Glycyl-Prolyl-Glutamate or GPE), we find that use of cG-2-AllylP hasdistinct and substantial advantages over other pharmacological agents,including GPE.

For example, cG-2-AllylP need not be delivered intravenously,subcutaneously, intraventricularly, or parenterally. In fact, oralformulations comprising micro-emulsions, coarse emulsions, liquidcrystal preparations, nanocapsules and hydrogels can be used inmanufacture of orally administered preparations such as tablets,capsules and gels that can improve neurological function and treatneurodegenerative conditions. Compounds of this invention can be used insituations in which a patient's motor functioning is below that neededto swallow a table or capsule. There are several types of soluble gelsfor oral administration of compounds, and these can be used to deliver acompound or composition of this invention to a patient. BecausecG-2-AllylP can be easily administered orally and is orally effective intreating neurodegenerative disorders, including Rett Syndrome, weconclude that cG-2-AllylP can be convenient and beneficial for long-termtherapy of patients with Rett Syndrome.

Further, because Rett Syndrome shares key features with other autismspectrum disorders, compounds of this invention can be useful inproviding therapeutic benefit from animals having other ASD, and inhumans with autism, Asperger Syndrome, Childhood DisintegrativeDisorder, and Pervasive Developmental Disorder-Not Otherwise Specified(PDD-NOS).

Example 24: Treatment of ASDs

There are several animal systems that have been used to evaluatetherapeutic efficacy of compounds in ASDs.

Shank3-Deficient Mouse Model

Shank3-deficient mice are used in the study as a model of 22q13 deletionsyndrome associated with ASD.

22q13 deletion syndrome has been linked with deletions or mutations inShank3 gene (Bonaglia et al, 2006). The Shank3 gene codes for a masterscaffolding protein which forms the framework in glutamatergic synapses(Boeckers et al, 2006). Shank3 is a crucial part of the core of thepostsynaptic density (PSD) and recruits many key functional elements tothe PSD and to the synapse, including components of thea-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA),metabotropic glutamate (mGlu), and N-methyl-D-aspartic acid (NMDA)glutamate receptors, as well as cytoskeletal dements. Recent studiesexploring the rate of 22q13 deletions/Shank3 mutations suggest thathaploinsufficiency of Shank3 can cause a monogenic form of ASD with afrequency of 0.3% to 1% of ASD cases (Durand et al, 2007; Moessner etal, 2007; Gauthier et al, 2008).

The generation of the mouse model with disrupted expression offull-length Shank3 has been previously described in the art (Bozdagi etal., Molecular Autism 2010, 1:13, p 4). Briefly, Bruce4 C37BL/6embryonic stem cells were used to generate a mouse line that had loxPsites inserted before exon 4 and exon 9. The floxed allele was excisedand a line was maintained with a deletion of exons 4 to 9, i.e. acomplete deletion of the ankyrin repeat domains of Shank3. Wild-type(+/+), heterozygous (+/−) and knockout (−/−) mice were produced, withMendelian frequencies from heterozygote-heterozygote crosses. A 30%reduction of full length Shank3 mRNA was confirmed in heterozygotes(qPCR) as well as a reduced expression of Shank3 protein (byimmunoblotting with Skank3 antibody N69/46).

Heterozygous mice generated by crossing wild-type mice withheterozygotes are used in this example to best model thehaploinsufficiency of Shank3, responsible for 22q13 deletion syndrome.

Methods

Drug Treatment

1 to 3 month old wild-type and heterozygous Shank3-deficient mice aredivided into 4 treatment groups: placebo treated wild-type, placebotreated Shank3-deficient group and two Shank3-deficient cG-2-AllylPtreated groups. The animals are given placebo (water) or cG-2-AllylPformulated in water administered orally, b.i.d for 14 days. cG-2-AllylPis administered at two doses: 15 or 60 mg/kg.

Methodology

A detailed description of the methodology can be found in Bozdagi et al.(Molecular Autism 2010, 1:15).

Behavioral Analyses

Behavioral assessments are made at several time points, and includeanalysis of social interactions and ultrasonic social communication, inline with the methodology described by Bozdagi et al. Briefly,male-female social interactions in each treatment group are evaluated.The subject males are group-housed and individually tested in cleancages with clean litter. Each testing session lasts 5 min. Each of thesubject mice is paired with a different unfamiliar estrus C57BL/6Jfemale. A digital closed circuit television camera (Panasonic, Secaucus,N.J., USA) is positioned horizontally 30 cm from the cage. An ultrasonicmicrophone (Avisoft UltraSoundGate condenser microphone capsule CM 15;Avisoft Bioacoustics, Berlin, Germany) is mounted 20 cm above the cage.Sampling frequency for the microphone is 250 kHz, and the resolution is16 bits. While the equipment used cannot distinguish between callsemitted by the male subject and female partner, the preponderance ofcalls during male-female interactions in mice is usually emitted by themale. The entire apparatus is contained in a sound-attenuatingenvironmental chamber (ENV-018V; Med Associates, St Albans, Vt., USA)illuminated by a single 25-Watt red light. Videos from the male subjectsare subsequently scored by an investigator uninformed of the subject'sgenotype and treatment group on measures of nose-to-nose sniffing,nose-to-anogenital sniffing and sniffing of other body regions, usingNoldus Observer software (Noldus Information Technology, Leesburg, Va.,USA). Ultrasonic vocalizations are identified manually by two highlytrained investigators blinded to genotype/treatment group information,and summary statistics are calculated using the Avisoft package.Interrater reliability is 95%. Data are analysed using an unpairedStudent's t-test.

Olfactory habituation/dishabituation testing is conducted in male andfemale mice for each group. The methodology is as previously described(Silverman et al 2010, Yang et al 2009 and Silverman et al 2010).Non-social and social odors are presented on a series of cotton swabsinserted into the home cage sequentially, each for 2 min, in thefollowing order; water, water, water (distilled water); almond, almond,almond (1:100 dilution almond extract); banana, banana, banana (1:100dilution artificial banana flavouring); social 1, social 1, social 1(swiped from the bottom of a cage housing unfamiliar sex-matched B6mice); and social 2, social 2, social 2 (swiped from the bottom of asecond cage housing a different group of unfamiliar sex-matched129/SvImJ mice). One-way repeated measures ANOVA is performed withineach treatment group for each set of habituation events and eachdishabituation event, followed by a Tukey post hoc test.

Hippocampal Slice Electrophysiology

Post-mortem, acute hippocampal slices (350 μm) are prepared from miceusing a tissue chopper. Slices are maintained and experiments areconducted at 32° C. Slices are perfused with Ringer's solutioncontaining (in mM): NaCl, 125.0; KCl, 2.5; MgSO₄, 1.3; NaH₂PO₄, 1.0;NaHCO₃, 26.2; CaCl₂, 2.5; glucose, 11.0. The Ringer's solution isbubbled with 95% 02/5% CO2, at 32° C., during extracellular recordings(electrode solution: 3 M NaCl). Slices are maintained for 1 hr prior toestablishment of a baseline of field excitatory postsynaptic potentials(fEPSPs) recorded from stratum radiatum in area CA1, evoked bystimulation of the Schaffer collateral-commissural afferents (100 μspulses every 30 s) with bipolar tungsten electrodes placed into areaCA3. Test stimulus intensity is adjusted to obtain fEPSPs withamplitudes that are one-half of the maximal response. The EPSP initialslope (mV/ms) is determined from the average waveform of fourconsecutive responses. Input-output (I/O) curves are generated byplotting the fEPSP slope versus fiber volley amplitude in low-Mg²⁺ (0.1mM) solution. AMPA receptor-mediated and NMDA receptor-mediated I/Orelationships are measured in the presence of ionotropic glutamatereceptor antagonists: 2-amino-2-phosphonopentanoic acid APV (50 μM) and6-cyano-7-nitroquinoxaline-2,3-dione CNQX (100 μM). Paired-pulseresponses are measured with interstimulus intervals of 10 to 200 ms, andare expressed as the ratio of the average responses to the secondstimulation pulse to the first stimulation pulse.

LTP is induced either by a high-frequency stimulus (four trains of 100Hz, 1 s stimulation separated by 5 min), or by theta-burst stimulation(TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms), or by asingle 100 Hz stimulation, for control and genetically-modified mice. Toinduce long-term depression (LTD), Schaffer collaterals are stimulatedby a low frequency or paired-pulse low frequency stimulus (900 pulses at1 Hz for 13 min) to induce mGlu receptor-dependent LTD. Data areexpressed as means±SD, and statistical analyses are performed usinganalysis of variance (ANOVA) or student's t-test, with significance setat an a level of 0.03.

Results

Behavioral

Cumulative duration of total social sniffing by the male test subjectsis lower in placebo treated Shank3-deficient group than in placebotreated wild-type group. In addition, fewer ultrasonic vocalizations areemitted by the placebo treated Shank3-deficient group than by thewild-type controls during the male-female social interactions.

cG-2-AllylP treatment in the two Shank3-deficient groups results in asignificant increase in the cumulative duration of total social sniffingin comparison to the placebo treated Shank3-deficient group. Moreover,the cG-2-AllylP treated groups display an increased number of ultrasonicvocalizations than the placebo treated mutant group.

In the olfactory habituation/dishabituation study, intended to confirmthat the mice are able to detect social pheromones, all 4 groups displaynormal levels of habituation (indicated by decreased time spent insniffing the sequence of three same odors), and the expecteddishabituation (indicated by increased spent in sniffing the differentodor).

Electrophysiology

Plotting field excitatory postsynaptic potential (fEPSP) slope versusstimulus intensity demonstrates a reduction in the I/O curves in theplacebo treated Shank3-deficient group versus the control group. In theheterozygous placebo treated group we also observe a decrease in AMPAreceptor-mediated field potentials, reflected in a 30% decrease in theaverage slope of I/O function compared to the wild-type control group.In contrast, when the I/O relationship is analyzed in the presence ofthe competitive AMPA/kianate receptor antagonist CNQX to measuresynaptic NMDA receptor function, there is no difference between thewild-type and placebo treated heterozygous groups. These resultsindicate that there is a specific reduction in AMPA receptor-mediatedbasal transmission in the Shank3 heterozygous mice.

cG-2-AllylP treatment in both heterozygous groups normalizes the AMPAreceptor-mediated field potentials and causes an increase in the averageslope of I/O function compared to the placebo treated Shank3-deficientgroup.

The maintenance of LTP in the placebo treated Shank3-deficient group isclearly impaired in comparison to the wild-type control. TBS LTP tests(10 bursts of four pulses at 100 Hz separated by 200 ms) also show asignificant decrease in the potentiation at 60 min after TBS in theplacebo treated Shank3-deficient group. In contrast to the alteredsynaptic plasticity observed with LTP, long-term depression (LTD) wasnot significantly changed in the mutant group. cG-2-AllylP treatmentincreases hippocampal long-term potentiation (LTP) and its maintenancein both Shank3-deficient group in comparison to the placebo treatedShank3-deficient group.

Discussion and Conclusions

Poor social competencies and repetitive behaviors are the commonfeatures and key diagnostic measures of all forms of ASD. Delayedintellectual development and underdeveloped language skills are also acommon feature present in all ASD, excluding Asperger syndrome.

The animal models described above have been accepted in the art asdemonstrating similar symptoms to the clinical human conditions. Allmutant models discussed above (NLGN3, NLGN4, CADM1, NRXN1, FMR1, shank3)exhibit impaired social skills or increased social anxiety. Decreasedexcitatory transmission into the hippocampus has been identified inNRXN1, shank3, MeCP2 and FMR1 mutant animal models. At present nopolygenetic or multifactorial models of ASD have been described. Theanimal models described above, based on genetic defects that are knownto produce ASD in human population, provide the best opportunity to testthe efficacy of ASD therapies.

Therefore the efficacy of cG-2-AllylP in animal models of ASD isreasonably predictive of its efficacy in a human subject suffering fromASD.

Example 25: Measurement of the Signaling Proteins Phospho-ERK1/2 andPhospho-Akt by Phospho-Flow Cytometry Analysis of FXS Mice Lymphocytes

Signal transduction pathways link external stimuli with cellularresponses, which normally regulate cell proliferation, death, anddifferentiation. We use phospho-specific antibodies for ERK1/2 and Aktthat recognize these proteins only when they are phosphorylated. One ofthe unique features of flow cytometry is its ability to performmeasurements of phosphorylation states at the that is not obtained bystandard biochemical techniques this clearly has wide potential forstudying drug treatment effect.

Methods

Lymphocytes are isolated from five fmr1-knockout and five wild-typetitter mate control mice, per study time after injection withcG-2-AllylP. Lymphocytes are examined for activation of two signalingeffectors, p-ERK1/2 and p-Akt using phosphorylation status as a measureof activation by flow cytometry (phospho-flow).

Measures taken are:

1. Flow cytometry total and phosphorylated ERK in lymphocytes

2. Flow cytometry total and phosphorylated AKT in lymphocytes

Results

We find that lymphocytes isolated from fmr1-knockout mice exhibitactivation of ERK1/2 and Akt phospho-epitopes. The mean fluorescentintensity (MFI) levels for p-AKT and p-ERK1/2 in fmr1-knockout micetreated with cG-2-AllylP decreases at all time points: 15, 30, 60 and240 minutes after a single treatment. We find similar reductions of MFIin fmr1-knockout mice after 5 and consecutive days of cG-2-AllylPtreatment.

In summary, cG-2-AllylP produces a significant reduction in phosphoactivation of ERK1/2 and Akt in fmr1-knockout mice. This resultindicates that p-ERK and p-Akt are useful biological markers to assesstherapeutic efficacy in treating human beings with ASD or NDD. Becauseexpression of pERK and pAkt in lymphocytes are similar to expression ofthose phosphorylated proteins in brains of fmr1-knockout mice,observation of therapeutic effects in lymphocytes is reasonablypredictive of effects of cG-2-AllylP in the brains of affected animals,including human beings.

Example 26: Treatment of Human Beings Having Fragile X Syndrome UsingG-2-AllylP

To determine whether cG-2-AllylP is effective in treating patients withfragile X Syndrome, we carry out a double-blinded, placebo-controlledstudy.

Methods

Patients

Male patients having Fragile X Syndrome are diagnosed by geneticanalysis demonstrating full fmr1 mutation. Symptoms are evaluated usingone or more of the clinical evaluative tools discussed herein above.Each patient is scored according to one or more clinical evaluative toolfor one or more of Repetitive Behavior (RBB), Anxiety, and Sociability.A Clinical Global Impression-Severity (CGI-S) score of 4 or greater, oran ABC total score of 30 or greater are enrolled.

Drug Delivery

The enrolled patients are divided into groups. All enrollees commencethe study with a 2-week single-blind administration of placebo b.i.d.Thereafter, cG-2-AllylP is administered orally at a dose of 35 mg/kgb.i.d. (n=20) or 70 mg/kg b.i.d (n=20) for 28 days, followed by 28 daysof placebo, or if randomized to placebo, cG-2-AllylP is administered ata dose of 70 mg/kg b.i.d. starting on day 42 and ceasing on day 70.Prior to dispensing study medication, cG-2-AllylP is reconstituted witha strawberry flavored diluent to provide a liquid for oraladministration. Placebo (n=20) is strawberry diluent and water.

Assessments

Pharmacokinetics

Blood samples from all subjects in each group are taken at Day 42 andDay 70. Four (4) samples are collected commencing on Day 42 (pre-doseand 2-4 hours post dose) and Day 70 (pre-dose and 2-4 hours post dose).A back-up sample for each time point is also collected.

Efficacy

Efficacy is determined using Clinical Global Impression of Severity(CGI-S), Clinical Global Impression of Improvement (CGI-I), Fragile XSyndrome Rating Scale, clinician-completed Fragile X Domain SpecificConcerns (Visual Analog Scale), Caregiver Top Three Concerns (VisualAnalogue Scale), Aberrant Behavior Checklist, Vineland Adaptive BehaviorScale, CASI-16, CYBOCS-PDD, seizure diary, computerized eye tracking,computerized measurement of cognition using the KiTap, and theExpressive Language Sampling Task.

Efficacy Outcome Measures

The following four groups of efficacy outcome measures are evaluated,comparing two dosage levels of cG-2-AllylP, separately and combined,with placebo.

Global Functional Outcome Measures

The following measures are assessed at Baseline, and during treatmentand the changes compared between active and placebo groups.

Changes in the Fragile X Syndrome Rating Scale are calculated for eachsubject between Baseline (pre-treatment) Day 14, and end of treatment(Days 42 and 70).

Global outcome is measured by the Clinical Global Impression-Severityand Improvement scales (CGI-S and CGI-I) at each clinic visit, fromBaseline onwards (e.g. Days 14, 28, 42, 56, 70, and 84.

Changes in the clinician-completed fragile X Domain Specific Concerns,as captured via a Visual Analog Scale (VAS), is calculated for eachsubject between Baseline (pre-treatment), Day 14, and end of treatment(Days 42 and 70).

Changes in Caregiver Top Three Concerns (related to the subject'sFragile X syndrome) as captured via a Visual Analogue Scale (VAS) iscalculated within subjects between Baseline (pre-treatment). Day 14, andend of treatment (Days 42 and 70).

Changes in the CASI-16, CYBOCS-PDD, Aberrant Behavior Checklist (ABC),Expressive Language Sampling Task and Vineland Adaptive Behavior Scales(VABS) is calculated for each subject between Baseline, Day 14, and Days42, and 70.

Physiological Outcome Measures

Serum levels and changes of standard hematology and chemistry parameters(including thyroid function) are calculated from Baseline through to Day70. Fundoscopy and tonsil size is documented at Baseline, and Days 14,28, 42, 36, 70 and 84. Flow cytometry is used to assess thephosphorylation status of the enzymes Akt and ERK in peripherallymphocytes, on blood samples obtained on Days 14, 28, 42, 36, and 70.ECG is assessed at Screening, Baseline, Days 28, 42, 36 and 70.

Cognitive/Automated Outcome Measures

The following measures are assessed at Baseline, during and post studydrug administration. Computerized measurement of cognition using theKiTap, is assessed at Baseline, Day 14, Day 42, Day 70 and Day 84.Computer-based eye tracking assessment is measured at Baseline, Day 14,28, 42, 36 and Day 70.

Pharmacokinetic-Pharmacodynamic Relationships

Matched pharmacokinetic (PK) and efficacy (PD) measures are collectedfrom all patients randomized to receive either 35 mg/kg or 70 mg/kg oralcG-2-AllylP twice daily. The pharmacodynamic markers include GlobalFunctional, Physiological and Cognitive/Automated Outcomes. The approachis to assess changes in efficacy measures over the course of the studyand to correlate these with measured or calculated pharmacokineticendpoints. In addition PK/PD models are used to establish a relationshipbetween blood concentration of cG-2-AllylP and effect, whereappropriate.

Statistical Methods

Efficacy

ANOVA, ANCOVA and Chi-square tests are used to compare efficacy measurelevels and changes between treatment groups. Each dose group is comparedwith the concurrent placebo group and the combined dose group iscompared with the combined placebo group. A two tailed p-value <0.05 isconsidered to indicate statistical significance.

A total sample size of 60 (40:20) enables effect sizes >0.80 betweenactive and placebois detected as statistically significant (2-tailedα=0.05) with 80% power on key outcome measures such as the Fragile XSyndrome Rating Scale. For comparisons within each dose group (i.e.comparison of assessment times), effect sizes >0.7 within active andplacebo are detected as statistically significant (2-tailed α=0.05) with80% power.

Pharmacokinetics

The maximal concentration (Cmax; peak), minimum concentration (Cmin;trough), C0-4 and area under the curve (AUC) parameters at steady stateare calculated directly from the cG-2-AllylP concentration data andsummarized at each time point using standard descriptive statisticsincluding means, medians, geometric means, standard deviations, ranges,and 95% confidence intervals.

In order to determine whether there are associations between PKparameters and efficacy measures, the changes in efficacy measures overthe course of the study are correlated with PK parameters estimated atthe different time points. These associations are statistically testedusing correlation coefficients and general linear models.

Results and Conclusion

We find that cG-2-AllylP is well tolerated by the patients. We furtherfind that cG-2-AllylP has clinically relevant and significant effects toimprove clinical outcomes as measured using one or more evaluative toolsdescribed herein. We also find that cG-2-AllylP normalizes pERK and pAktlevels.

This study demonstrates that of cG-2-AllylP is effective in treatingadverse symptoms of Fragile X Syndrome in human beings.

Example 26: Effects of cG-2-AllylP on Synaptic Plasticity FollowingPenetrating Ballistic-Like Brain Injury in Rats I

In this Example, we investigated whether cG-2-AllylP hasanti-inflammatory and anti-apoptotic activity after PenetratingBallistic-Like Brain Injury (PBBI). Molecules structured similarly tocG-2-AllylP have been shown to have memory enhancing effects or improvepassive-avoidance learning in vivo and promote neurite outgrowth invitro. This suggests that treatment with c(GP) analogues enhancesneuroplasticity and synapse formation. We therefore investigated whethercG-2-AllylP regulates genes and encoded proteins that govern synapticplasticity following PBBI.

Methods

Penetrating Ballistic-Like Brain Injury (PBBI)

The unilateral frontal PBBI model, which mimics the ballistic dynamicsof a bullet or fragment wound to the head (Williams, 2003; Williams,2006), was induced by stereotactic insertion of a custom probe throughthe right frontal cortex. A temporary cavity was formed by the rapidinflation/deflation (i.e. <40 msec) of an elastic balloon attached tothe end of the probe. The PBBI apparatus consisted of acomputer-controlled hydraulic pressure generator (Mitre Corp McLean,Va.), a PBBI probe and a stereotaxic frame equipped with acustom-designed probe holder as previously described (Lu, 2009;Williams, 2003). The injury severity was determined by the size of theballoon under control of the computerized hydraulic pressure. Theballoon diameter calibrated to 0.63 cm expansion represented 10% of thetotal rat brain volume thus expressing a 10% PBBI. All surgeries weredone under anaesthesia. Groups tested: Sham+vehicle, PBBI+vehicle,PBBI+cG-2-AllylP (30 mg/kg by oral gavage, 30 min post-injury, and againonce daily until endpoint). AU procedures were approved by theInstitutional Animal Care and Use Committee of WRAIR. Animals werehoused in a facility accredited by the AAALAC.

Oral Administration of cG-2-AllylP

cG-2-AllylP (or vehicle) was administered to the animals via oral gavageat a dose of 30 mg/kg.

ELISA

Target proteins were quantified with interleukin 1 beta (“IL-1beta”)(GenWay Biotech GWB-SKR107) and interleukin-6 (“IL-6”) (GenWay BiotechGWB-ZZD100) ELISAs according to manufacturer's instructions. Levels werecalculated and normalized to total protein concentration as determinedby BCA assay (n=9-10 per group).

Western Blotting

Samples were analysed at 24 hr and 3 and 7 days post-injury for allgroups (sham+vehicle, PBBI+vehicle, PBBI+cG-2-AllylP). Tissue washomogenized in RIPA buffer containing HALT protease inhibitors. Totalprotein was based on BCA assay. Blots were blocked 5% milk, probed withanti-ATF3, anti-BAX, or anti-BCL2. Blots were re-probed withanti-beta-actin antibody to control for protein loading. Analysis ofband intensity was done using an LAS4000 and ImageQuantTL software (GEHealthcare) (n=9-10 per group).

Neuroplasticity mRNA Arrays

cDNA was generated. From total RNA from individual animals using randomprimers. cDNA was plated unto a targeted PCR array produced by SABiosciences and product was detected using SyBr green fluorescence. Ctlevels were normalized to beta actin. Injury results were compared tosham to evaluate relative quantities (RQ) (n=6 per group).

Results

PBBI injury alone led to an acute (at 24 hrs. after PBBI) increase inthe inflammatory and apoptotic measures studied. FIG. 33A-33F showsthese results. PBBI increased the amount of IL-1beta (FIG. 33A) and IL-6(FIG. 33D). cG-2-AllylP decreased proinflammatory cytokines IL1-beta(FIG. 33C) and IL-6 (FIGS. 33D and 33F). ANOVA: * p<0.05, ** p<0.01;Error bar: SEM. cG-2-AllylP increased IL1-beta levels at 3 days afterPBBI, which may be compensatory for its downstream target IL-6.Treatment with cG-2-AllylP decreased levels of IL1-beta at 7 days afterPBBI (FIG. 33C). cG-2-AllylP treatment decreased IL-6 levels at both 24hrs. (FIG. 33D). and 7 days (FIG. 33E) after PBBI, but had little effectat 3 days (FIG. 33B). These results indicate that cG-2-AllylP reducedthese inflammatory cytokines.

FIG. 34 shows that PBBI significantly and substantially increasedexpression of BAX (FIGS. 34 A, 34B, and 34C) and BCL2 (FIGS. 34D, 34E,34F, 34G, and 34H). cG-2-AllylP did not significantly alter BAX (FIGS.34A-34C) or BCL2 (FIGS. 34D-34H) levels.

FIG. 35 shows that PBBI increased expression of ATF3 at all time points(FIGS. 35A-35C). Surprisingly, we found that cG-2-AllylP decreased ATF3at 24 hrs. after PBBI as measured globally by Western blotting (FIG.35A).

FIG. 36 shows that after PBBI, cG-2-AllylP treatment significantlyincreased Gria 4 (an AMPA receptor, FIG. 36F) at the 24 hr. time point.In addition, we observed trends including: decreased Crem (a CREBinhibitor, FIG. 36D), decreased NTF3 (FIG. 36J), decreased NTF4 (FIG.36K), decreased Pcdh8 (a tumor suppressor gene; FIG. 36L), decreasedBDNF (FIG. 36A), decreased Pim1; FIG. 36M) and increased Ppp3ca (FIG.36N).

Conclusions

These trends in RNA expression, particularly the increased Gria 4,decreased Crem, and decreased Pcdh8 expression promote neuroplasticity.The decreased NTF3 and NTF4 allows synaptic formation.

Collectively, these results indicate that cG-2-AllylP hasanti-inflammatory effects following severe PBBI and enhancedneuroplasticity. Because the results obtained in this experimentalsystem are reasonably predictive of effects seen in human beings,cG-2-AllylP and similar cyclic GP compounds can be effective in treatingsymptoms of mild, moderate, or severe traumatic brain injury.

Example 27: Effects of cG-2-AllylP on Synaptic Plasticity FollowingPenetrating Ballistic-Like Brain Injury in Rats II: Growth-AssociatedProtein 43 and Synaptophysin

In this Example, we examined the role of cG-2-AllylP on expression ofgenes and proteins related involved in neuroplasticity followingpenetrating ballistic-like brain injury (PBBI; 10% injury severity) inrats.

Methods

The methods for producing PBBI in this Example is the same as describedabove for Example 26. Adult Sprague-Dawley rats were randomly assignedinto three groups: sham (craniotomy only), PBBI+vehicle (i.e. H₂O), andPBBI+cG-2-AllylP. cG-2-AllylP (or vehicle) was administered via oralgavage at 30 mg/kg at 30 min post-injury and continued once dailythereafter for 7, 14 or 28 days. At each treatment endpoint, rats wereperfused and brains were processed for histological analysis(n=5-6/group/time-point). For detection of axonal sprouting,immunohistochemical detection of growth-associated protein-43 (GAP-43)was employed. Synaptogenesis was determined by immunohistochemistry forsynaptophysin (SYN). For histological quantification, the integrateddensity in the hippocampal region was determined using NIH ImageJsoftware.

Results

In the vehicle treatment group, PBBI significantly decreased GAP-43expression in the ipsilateral hippocampus at 7d, 14d and 28dpost-injury, and in the contralateral hippocampus at 7d and 14dpost-injury (p<0.05 vs. sham). Significant reductions in SYN stainingwere detected at 14d and 28d post-injury in the ipsilateral hippocampusand at 14d post-injury in the contralateral hippocampus in thePBBI+vehicle group (p<0.03 vs. sham). Continuous treatment withcG-2-AllylP showed no effect on injury-induced reductions in GAP-43 orSYN expression at 7d or 14d post-PBBI. However, at 28 days post-injury,cG-2-AllylP treatment attenuated PBBI-induced reductions in both GAP-43and SYN expression to levels that did not differ significantly from shamcontrols, indicative of an intermediate treatment effect.

CONCLUSIONS

Histological analysis indicates that PBBI induced significant reductionof axonal sprouting and synaptogenesis during sub-acute to chronic phaseafter injury. These results show a trend of cG-2-AllylP in promotingneuroplasticity. Because the animal system used is predictive ofneuroplasticity in human beings, these results indicate that cG-2-AllylPcan be effective in ameliorating adverse effects of brain injury.

The descriptions and examples provided herein are for purposes ofillustration only. The scope of this invention to is not intended to belimited to the described embodiments. Other embodiments incorporatingelements of the invention can be practiced without undue experimentationby persons of ordinary skill in the art. All such embodiments aretherefore considered to be part of this invention.

REFERENCES

The following references, and all patents, patent applications and otherpublications cited herein are incorporated fully by reference as ifseparately so incorporated.

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We claim:
 1. A method for treating one or more symptoms in a mammalhaving autism, comprising administering to the mammal, apharmaceutically effective amount of a compound comprising cyclicGlycyl-2-Allyl Proline (cG-2-AllylP), cyclic cyclohexyl-G-2MeP, orcyclic cyclopentyl-G-2MeP.
 2. The method of claim 1, wherein saidcompound is formulated in a solution, in a gel, along with one or moreexcipients, carriers, additives, adjuvants, binders, in a tablet, in acapsule, in a nanocapsule, in a microemulsion, in a coarse emulsion, orin a liquid crystal.
 3. The method of claim 1, where the compound isadministered either directly or indirectly via the circulation.
 4. Themethod of claim 1, where said compound is administered via a routeselected from the group consisting of oral, intraperitoneal,intravascular, peripheral circulation, subcutaneous, intraorbital,ophthalmic, intraspinal, intracisternal, topical, infusion, implant,aerosol, inhalation, scarification, intracapsular, intramuscular,intranasal, buccal, transdermal, pulmonary, rectal, and vaginal.
 5. Themethod of claim 1, where said effective amount has a lower limit ofabout 0.001 milligrams per kilogram mass (mg/kg) of the mammal and anupper limit of about 100 mg/kg.
 6. The method of claim 1, wherein saidtreatment produces an improvement in a symptom of the disorder asassessed using one or more clinical tests selected from the groupconsisting of Aberrant Behavior Checklist Community Edition (ABC),Vineland Adaptive Behavior Scales, Clinical Global Impression ofSeverity (CGI-S), Clinical Global Impression Improvement (CGI-I), theCaregiver Strain Questionnaire (CSQ), electroencephalogram (EEG) spikefrequency, overall power in frequency bands of an EEG, hemisphericcoherence of EEG frequencies, stereotypic hand movement, eye tracking,QTc variability, heart rate variability (HRV), respiratoryirregularities, and abnormal coupling of cardiac and respiratoryfunction compared to control animals not suffering from said disorder.7. The method of claim 1, where said treatment reduces one or moresymptoms selected from the group consisting of anxiety, impairment insocial interaction, impairment in the use of multiple nonverbalbehaviors, failure to develop peer relationships appropriate todevelopmental level, lack of spontaneous seeking to share enjoyment,interests, or achievements, lack of social or emotional reciprocity,impairment in communication, restricted and repetitive interests andbehaviors, lack of spontaneous make-believe play, abnormal fearconditioning, abnormal social behaviour, repetitive behaviour, abnormalnocturnal behaviour, seizure activity, abnormal locomotion, abnormalexpression of Phospho-ERK1/2, abnormal expression of Phospho-Akt, andbradycardia.
 8. A method for treating a symptom of autism in a mammal,comprising administering to the mammal, a compound having the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein X¹ isselected from the group consisting of NR′, O and S; X² is selected fromthe group consisting of CH₂, NR′, O and S; R¹, R², R³, R⁴ and R⁵ areindependently selected from the group consisting of —H, —OR′, —SR′,—NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl; or R⁴ and R⁵ taken togetherare —CH₂—(CH₂)_(n)-CH₂— where n is an integer from 0-6; or R² and R³taken together are —CH₂—(CH₂)_(n)—CH₂— where n is an integer from 0-6;with the proviso that when R¹=methyl and R²=R³=R⁴=H then R⁵ # benzyland; when R¹=H, at least one of R² and R³≠H.
 9. The method of claim 8where R¹=methyl.
 10. The method of claim 8 where R¹=allyl.
 11. Themethod of claim 8 where R²=R³=methyl and X²=S.
 12. The method of claim 8where R¹=allyl, R²=R³=R⁴=R=H, X¹=NH, X²=CH₂.
 13. The method of claim 8where R¹=methyl, R²=R³=H, R⁴ and R⁵ taken together are —CH₂—(CH₂)₃—CH₂—,X¹=NH, X²=CH₂.
 14. The method of claim 8 where R¹=methyl, R²=R³=H, R⁴and R⁵ taken together are —CH₂—(CH₂)₂—CH₂—, X¹=NH, X²=CH₂.
 15. Themethod of claim 8, wherein said compound is cG-2-AllylP.
 16. The methodof claim 8, wherein said compound is cyclic cyclohexyl-G-2MeP.
 17. Themethod of claim 8, wherein said compound is or cycliccyclopentyl-G-2MeP.
 18. The method of claim 8, said compound furthercomprising one or more pharmaceutically acceptable excipients,additives, carriers, or adjuvants in solution.
 19. The method of claim8, said compound further comprising one or more excipients, carriers,additives, adjuvants or binders in a tablet.
 20. The method of claim 8,said compound further comprising a microemulsion, coarse emulsion, orliquid crystal in a capsule.
 21. The method of claim 8, where the methodfurther comprises administering said compound along with apharmaceutically acceptable excipient, or in a gel.
 22. The method ofclaim 8, where the method further comprises administering said compoundalong with a pharmaceutically acceptable excipient and a binder.
 23. Themethod of claim 8, where the method further comprises administering saidcompound along with a pharmaceutically acceptable excipient and acapsule.
 24. The method of claim 8, where said treatment reduces one ormore symptoms selected from the group consisting of anxiety, impairmentin social interaction, impairment in the use of multiple nonverbalbehaviors, failure to develop peer relationships appropriate todevelopmental level, lack of spontaneous seeking to share enjoyment,interests, or achievements, lack of social or emotional reciprocity,impairment in communication, restricted and repetitive interests andbehaviors, lack of spontaneous make-believe play, abnormal fearconditioning, abnormal social behaviour, repetitive behaviour, abnormalnocturnal behaviour, seizure activity, abnormal locomotion, abnormalexpression of Phospho-ERK1/2, abnormal expression of Phospho-Akt, andbradycardia.